Aqueous Systems For The Preparation Of Lipid Based Pharmaceutical Compounds; Compositions, Methods, And Uses Thereof

ABSTRACT

The present invention relates to a methods of preparing active compounds complexed with lipids using aqueous systems that are free of organic solvents, and methods of using the complexes, e.g., in treating a disease in a subject. In some embodiments, the present invention comprises a composition comprising a complex comprising at least one active compound, e.g., a polyene antibiotic, an immunosuppressant agent such as tacrolimus or a taxane or taxane derivative, and one or more lipids. In some embodiments, the present invention provides a method comprising preparing a composition comprising a lipid complex comprising at least one active compound and at least one lipid and administering the composition to a subject. In certain embodiments the subject is a mammal. In certain preferred embodiments, the subject is human.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims is a continuation of U.S. patent applicationSer. No. 11/915,345, filed Nov. 24, 2009, which is a §371 national entryapplication of international patent application PCT/US2007/080984, filedOct. 10, 2007, which priority to both U.S. Provisional Application60/850,446, filed Oct. 10, 2006, and U.S. Provisional Application60/957,022, filed Aug. 21, 2007 each of which is incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates to compositions comprising active components orcompounds, e.g., pharmaceutical compounds, and lipids, including, e.g.,complexes, micelles, emulsions, liposomes or lipidic particle, andmixture of micelles and vesicles. The invention further relates to theirmethods of preparation, and uses in the treatment of diseases. By way ofexample and not by way of limitation to any particular active component,in some embodiments, the invention relates to compositions comprisingamphotericin B, with or without deoxycholate, and one or more lipids,their methods of preparation in an aqueous system, and their uses forthe treatment of diseases, such as mammalian diseases. In someembodiments, the invention relates to compositions comprising, e.g.,immunosuppressants such as tacrolimus, anticancer compounds such asdocetaxel or paclitaxel, or any other compound of the taxane family, andone or more lipids, their methods of preparation in the absence oforganic solvents, and their uses for treatment, e.g., of mammaliandiseases. Methods according to the present invention are suitable forpractice on an industrial manufacturing scale, and may be practiced,e.g., as a continuous process. Other significant advantages of thesemethods include simplicity, speed of particle formation, ease of scalingto large volumes, the formation of lipid suspensions of highconcentration and defined particle size and the ability to aqueoussystems in the encapsulation of pharmaceutically active compounds havingpoor water solubility.

BACKGROUND OF THE INVENTION

Most lipidic preparation systems involve the use of organic solventssuch as dimethylsulfoxide, dimethylformamide, methylene chloride,chloroform, ethanol or methanol. Organic solvents can pose health risks,e.g., for production workers, and removal of organic solvents isgenerally a cumbersome process. Hence there is a need for processes forpreparation of lipid based formulations without the need for fromorganic solvents.

Polyene antibiotics provide one example a class of active pharmaceuticalcompounds having limited solubility in aqueous systems. Polyeneantibiotics are widely used in the treatment of both pre-systemic andsystemic fungal infections. They are produced by several differentspecies of Streptomyces. Recent interest in these antibiotics isstimulated due to their synergistic antifungal action with other agents(Medoff, G. and Kobayashi, G. S. 1975) and by reports of antitumoraction (Valeriote, F. et al. 1976). In particular, polyene antibioticssuch as amphotericin B (AmB) and Nystatin (Nys) have remained the mosteffective and widely used agents in the treatment of fungal infections.In addition several, but not all, of these agents have been shown tohave immunoadjuvant properties (Hammarstron, L. and Smith, C. I. E.,1977; Little, J. R. et al. 1978).

The polyene antibiotics target sterols, specifically ergosterol, whichis the abundant and main sterol of fungal membranes. The different typesof polyene antibiotics display different modes of action, despite thatthey share a common target. The larger polyenes like amphotericin andnystatin form together with ergosterol pore structures in the plasmamembrane which collapse vital ion gradients, thereby killing the cells.The smaller uncharged filipin also destroys the membrane barrier, but bya completely different mechanism. Filipin forms large complexes withsterols between the leaflets of the lipid bilayer, resulting in breakageof the membrane (De Kruijff and Demel, 1974). Natamycin like the otherpolyene-antibiotics specifically binds to ergosterol in the membrane,but this does not result in a loss of barrier function.

Amphotericin B is a parental antifungal antibiotic produced as afermentation by-product of streptomyces nodusus, a soil actinomycete. Itbinds to sterols in the cell membranes of both fungal and mammaliancell. It is usually fungistatic in vivo but can have fungicidal activityat high concentrations or against extremely susceptible organisms. Itshigher affinity for ergosterol, the sterol found in fungal cellmembranes, over cholesterol, the sterol found in human cell membranes,allows amphotericin B to be used systematically. As a result of thisbinding, fungal membrane integrity is impaired, causing the loss ofintracellular potassium and other cellular contents. Some adversereactions to amphotericin B, such as electrolyte loss andnephrotoxicity, are an extension of its pharmacologic action, whileanaphylactoid infusion-related reactions may be related to stimulationand release of prostaglandin synthesis. Anemia may be secondary to aninhibition of erythropoietin production.

Amphotericin B is widely used for severe life-threatening fungalinfections. Its use limited by a dose-dependent nephrotoxicity,manifested by a reduction in glomerular filtration rate and tubulardysfunction. An elevated excretion of creatinine associated withamphotericin B is not only a marker for renal dysfunction but is alsolinked to a substantial risk for the use of hemodialysis and a highermortality rate; therefore, amphotericin B nephrotoxicity is not benigncomplication and its prevention is essential. (Deray, G. et al.Nephrologie, 2002).

Amphotericin B is poorly soluble in water, alcohols, chloroform, andother common halocarbon solvents. While amphotericin B is an effectivefungicide, it is dangerously toxic at concentrations slightly above thetherapeutic concentration. Encapsulation in liposomes appears to reducethe in vivo toxicity to mammalian cells, and leaving the fungicidalactivity relatively unaltered (F. C. Szoka et. al., 1987). Liposomeshave been used to encapsulate a large variety of compounds whichexhibits poor solubility or exhibits unacceptable toxicity attherapeutic dosages. The effects of liposome encapsulation oncytotoxicity and fungicidal activity of compounds such as amphotericin Bare dependent on the particular liposome structure (e.g., SUV, MLV etc.)and their method of preparation.

Development of new formulations using new lipid compositions is neededto improve the efficacy and to reduce the toxicity associated withcompositions such as polyene antibiotics, and particularly withamphotericin B, with or without deoxycholate.

Taxanes are a unique class of hydrophobic anticancer agents that exhibitcytotoxic activity by binding to tubulin and promoting inappropriatelystable, non-functional microtubule formation (Schiff P B et. al. 1979).Interference with microtubule function leads to disrupted mitosis andcell death. Certain taxanes, e.g., paclitaxel and docetaxel, areapproved for human use for the treatment of breast cancer, ovariancancer, non-small cell lung cancer and prostate cancer. The doselimiting toxicity profiles for these agents are somewhat different;paclitaxel has been most widely associated with peripheral neuropathiesand myalgias/athralgias, whereas docetaxel most commonly results influid retention that may be dose-limiting in some cases (Hennenfent, K.L et al 2006).

The taxanes, including but not limited to paclitaxel and docetaxel, arepractically insoluble in water and require a complex solvent system forcommercial formulation. Cremophor EL, a polyoxyethylated castor oilvehicle, and dehydrated ethanol USP (1:1, v/v) are used as solvents inthe commercial formulation of paclitaxel, while polysorbate 80 (Tween 80detergent) is employed in the formulation of docetaxel. Although thesesolvents systems are biologically and pharmacologically acceptable, theyhave known to have side effects, including acute hypersensitivityreactions and peripheral neuropathies. In addition, several reports havelinked these solvents to alterations in the pharmacokinetic profiles ofboth paclitaxel and docetaxel (ten Tije, A J et al. 2003).

Several formulations have been made to solublize the taxanes and tocircumvent the toxicities associated with it. All of these formulations,including lipid-based formulations (for example, liposomes), haverequired use of organic solvents to solubilize the active compoundduring the formulation process (Straubinger, et. al. U.S. Pat. No.5,415,868, 1995; Bisery, et al U.S. Pat. No. 6,146,663, 2000). As notedabove, the use of organic solvents results in a cumbersome process andhence an organic solvent-free formulation is needed to overcome theproblems associated with the existing formulations.

SUMMARY OF THE INVENTION

The present invention relates to new methods of preparing activecompounds complexed with lipids, and methods of using the complexes intreating a subject, e.g., for treating a disease in a subject. Thecomplex interaction may be ionic or lipophilic. In all the embodimentsof the present invention, the complex formation takes place in aqueousmedia. In some embodiments, the present invention comprises acomposition comprising a complex comprising at least one active agent,such as a polyene antibiotic, an immunosuppressant agent such astacrolimus or a taxane or taxane derivative and one or more lipids. Insome embodiments, the present invention comprises a method comprisingpreparing a composition comprising a complex comprising at least oneactive compound, e.g., a polyene antibiotic, and one or more lipids andadministering the composition to a subject. In certain embodiments thesubject is a mammal. In certain preferred embodiments, the subject ishuman.

An object of the present invention is to provide lipid formulations orcomplexes comprising at least one active component and at least onelipid, e.g., a phospholipid, formed without using organic solvent.

The amount of phospholipid included in a lipid complex according to thepresent invention is not limited to any particular amount or percentage(e.g., by weight) of the final composition or complex. In someembodiments, the proportion of the at least one phospholipid is betweenabout 5% to about 98% of a final lipid complex (e.g., a commerciallyusable form) by weight. In some preferred embodiments, the amount of theat least one phospholipid is between 10% to 90% of the lipid complex byweight.

In certain embodiments, a lipid formulation system according to thepresent invention has a pH of between about 4.0 and 8.0. In somepreferred embodiments, the pH is between about 4.5 and 7.5.

A lipid formulation of the present invention is not limited to anyparticular use or application. For example, a lipid formulation of anactive component according to the present invention comprising apharmaceutically active ingredient can be used for differentpharmaceutical applications. An aqueous system of the present inventioncan also be used in the formation of unloaded lipid complexes (e.g.,without any encapsulated active ingredient), for use, e.g., as controlsfor complexes comprising active components.

In some embodiments, the present invention comprises a compositioncomprising a complex comprising at least one anticancer agent and one ormore lipids. Examples of anticancer agents include but are not limitedto docetaxel, paclitaxel, epirubicin, endoxifen and the like.

As for example, it is possible to encapsulate or entrap tacrolimus, inthe inventive liposome system, such a pharmaceutical product is used,e.g., as an immunosuppressant or for the treatment of skin infection.Such a pharmaceutical product is particularly suitable for injection ororal usage. Furthermore, the known active ingredients are for thetreatment of cancer, liver disease, kidney diseases, AIDS, bacterial,fungal and viral infections.

In some embodiments, the present invention comprises a compositioncomprising a complex comprising at least one immunosuppressant agent andone or more lipids. Examples of immunosuppressant include but notlimited to tacrolimus and sacrolimus.

In some embodiments, the polyene antibiotic of a composition accordingto the present invention is amphotericin B with or without deoxycholate,while in some preferred embodiments; the amphotericin B deoxycholate isFUNGIZONE antibiotic. In some embodiments the amphotericin Bdeoxycholate is prepared from amphotericin B and sodium deoxycholate.

In some embodiments, the one or more lipids of a composition accordingto the present invention comprise one or more of cholesterol,cholesteryl sulfate and its salts (e.g., sodium salt), cholesterylhemisuccinate, cholesteryl succinate, cholesteryl oleate, polyethyleneglycol derivatives of cholesterol (cholesterol-PEG), coprostanol,cholestanol, cholestane, cholic acid, cortisol, corticosterone,hydrocortisone, or calciferol, while in some embodiments, the one ormore lipids comprises a sterol. In certain embodiments, the sterol isβ-sitosterol, stigmasterol, stigmastanol, lanosterol, α-spinasterol,lathosterol, campesterol or a mixture thereof.

In some embodiments, the one or more lipids of a composition accordingto the present invention comprises one or more of fatty acids having achain length of about C₄-C₃₄. In some embodiments, one or more fattyacid chains are unsaturated, while in some embodiments, one or more ofthe fatty acid chains are saturated. In some embodiments, one or more ofthe fatty acids are in salt form, while in some embodiments; one or moreof the fatty acids are in acidic form. In some embodiments, one or morefatty acids are in the form of an ester.

In some embodiments, one or more lipids of a composition according tothe present invention comprise a phospholipid. In some preferredembodiments, one or more of the lipids of the composition comprises aphosphatidylcholine or phosphatidylglycerol, while in some preferredembodiments; one or more of the lipids of the composition comprises aphosphatidylethanolamine, phosphatidylserine, phosphatdylinositol, orphosphatidic acid. In some preferred embodiments, one or more lipids ofthe present invention comprise a soybean phospholipid. In someparticularly preferred embodiments, a soybean phospholipid used in themethods and compositions of the present invention comprises a largeconcentration of phosphatidylcholine. In still more particularlypreferred embodiments, a soybean phospholipid used in the methods andcompositions of the present invention contains at least 90% by weightphosphatidylcholine. In some embodiments, one or more phospholipids arepegylated (PEG) derivatives of phospholipids. In certain embodiments,one or more of the lipids of the composition comprise a pegylatedderivative of a distearoylphosphatidylglycerol, adimyristoylphosphatidylglycerol, or a dioleoylphosphatidylglycerolphospholipid.

In some embodiments, one or more lipids of a composition according tothe present invention comprise a monoglyceride, a diglyceride, or atriglyceride lipid.

The method of composition, wherein said fatty acids of mono-, di-, andtriglycerides are selected from a group of saturated and unsaturatedfatty acids having short chain or long chain.

In some embodiments, one or more lipids of a composition according tothe present invention comprise a carbohydrate-based lipid. In certainpreferred embodiments, the one or more lipids of the compositioncomprise a galactolipid, mannolipid, and galactolecithin.

In some embodiments, a composition according to the present inventionfurther comprises polyethylene glycol (PEG). In some embodiments, thePEG has an average molecular weight ranging from 200-20,000, while incertain preferred embodiments, the average molecular weight of the PEGis in the range of 500-2000.

In some embodiments, a composition according to the present inventioncomprises active compound (for example amphotericin B, with or withoutsodium deoxycholate), cholesterol or cholesterol derivatives and one ormore phospholipids. In certain preferred embodiments, the compositioncomprises sodium deoxycholate, and the mole ratio of active compound(for example, amphotericin B) to sodium deoxycholate is about 1:2. Insome embodiments in which the composition comprises a cholesterolderivative, the cholesterol derivative is cholesteryl sulfate. In someembodiments wherein the phospholipid comprises soy phosphatidylcholineor hydrogenated phosphatidylcholine. In some preferred embodiments, themole ratio of active compound (for example, amphotericin B) andcholesterol or cholesterol derivative is in the range of about 1:1 and1:10, while in certain particularly preferred embodiments, the moleratio of active compound (for example, amphotericin B) and cholesterolor cholesterol derivative is in between about 1:1 and 1:5.

In some embodiments, one or more lipids of a composition according tothe present invention comprise hydrogenated soy phosphatidylcholine,wherein the mole ratio of active compound (for example, amphotericin B)and hydrogenated soy phosphatidylcholine is in between about 1:5 and1:80. In certain preferred embodiments, the mole ratio of activecompound (for example, amphotericin B) and hydrogenated soyphosphatidylcholine is in between about 1:5 and 1:60.

In some embodiments, a composition according to the present inventioncomprises active compound (for example, amphotericin B, with or withoutsodium deoxycholate) at a concentration of from about 0.5 mg/mL to about25 mg/mL while in some preferred embodiments, the active compound (forexample, amphotericin B with or without deoxycholate) of the compositionis at a concentration of from about 1 mg/mL to about 10 mg/mL. In someparticularly preferred embodiments, the composition of the inventioncomprises active compound (for example, amphotericin B, with or withoutdeoxycholate) is at a concentration of about 1 mg/mL to about 5 mg/mL.

In some embodiments, a composition according to the present inventioncomprises a total lipid concentration or proportion of from about 2.5%by weight to about 95% by weight, while in some preferred embodiments;the composition comprises a total lipid concentration of from about 5%by weight to about 95% by weight. In certain particularly preferredembodiments, the composition comprises a total lipid concentration offrom about 10% by weight to about 90% by weight.

In some embodiments, a composition according to the present inventioncomprises active compound (for example, amphotericin B), and totallipids including sodium deoxycholate (if used) having molar ratioranging from about 1:10 to about 1:100, while in some embodiments, themolar ratio is in between about 1:20 to about 1:70.

In some embodiments, a composition according to the present inventioncomprises active compound (for example, amphotericin B) and totallipid(s) including sodium deoxycholate having a weight-to-weight ratioranging from about 1:1 to about 1:100, while in certain preferredembodiments, the ratio is in between about 1:10 to about 1:60.

In some embodiments, a composition according to the present inventioncomprises a complex selected from the group consisting of a micelle andan emulsion. In certain preferred embodiments, the composition comprisesa plurality of micelles, wherein said micelles are in the form ofmonomeric, dimeric, polymeric or mixed micelles.

In some embodiments, a composition according to the present inventioncomprises complexes, liposomes, micelles, and/or vesicles that have adiameter of about 20 microns or less, while in some embodiments, thecomplexes, liposomes, micelles, and/or vesicles that have a diameter ofabout 10 microns or less. In some embodiments, the complexes, liposomes,micelles, and/or vesicles have a diameter of about 5 microns or less,while in some embodiments, the complexes, liposomes, micelles, and/orvesicles have a diameter of about 1 micron or less. In some embodiments,the complexes, liposomes, micelles, and/or vesicles have a diameter ofabout 500 nm or less, while in some embodiments, the complexes,liposomes, micelles, and/or vesicles have a diameter of about 200 nm orless. In some preferred embodiments, the complexes, liposomes, micelles,and/or vesicles have a diameter of about 100 nm or less.

The present invention is not limited to any particular form ofcomposition comprising the complex of the invention. For example, insome embodiments, a complex in a composition according to the presentinvention is in a lyophilized form. In some embodiments, the compositionfurther comprises a cryoprotectant. In certain preferred embodiments,the cryoprotectant comprises one or more sugars, while in particularlypreferred embodiments; the one or more sugars comprise trehalose,maltose, lactose, sucrose, glucose, and/or dextran.

In some embodiment of the methods and compositions of the presentinvention, the active ingredient is added after the preparation of theliposome system. In some particularly preferred embodiments, the activeingredient (e.g., an active pharmaceutical compound) is added to a lipidpreparation, e.g., a liposome system, immediately before use (e.g.,immediately before administration to a patient or subject). For example,in some embodiments, the active ingredient in dry form may be dispersedor emulsified into an aqueous unloaded liposome system, while in otherembodiments, a dried liposome system may be emulsified into water inwhich pharmaceutically active ingredient has been previously dispersedor emulsified. Pharmaceutical products prepared in this way show bettertransparency and may be easier to inspect, e.g., for the presence ofunwanted foreign particles.

In some embodiments, a complex in a composition according to the presentinvention is in a powder form, while in some embodiments, the complex isin a solution form. In some embodiments, the complex is in a suspensionform, while in other embodiments, the complex is in an emulsion form,while in still other embodiments, the complex is in a micelle form ormixed micellar form or in a liposome form. In some embodiments, thecomplex is in a lyophilized or gel form, while in some embodiments, thecomplex is in a paste form. In some embodiments, the complex is amixture of mixed micelles, liposomes or vesicles form.

In some embodiments, a composition according to the present invention isencapsulated in a capsule. In some preferred embodiments, the capsule isa gel capsule, while in some particularly preferred embodiments; thecapsule comprises an enteric coating.

In some embodiments, a complex in a composition according to the presentinvention is comprises a water insoluble, or poorly water soluble, drugthat is not a polyene antibiotic.

In some embodiments, a composition according to the present inventioncomprises an active component comprising a macrolide, e.g., Tacrolimus(Knoll, G. A. et al. 1999; Dumont F J In: Liebermann R, Mukherjee A,eds. 1996). or Sirolimus (Ingle G R, et al. 2000; Podder H, et al.2001). Macrolides such as Tacrolimus are currently used clinically forthe prophylaxis of liver and kidney transplant rejection. In someembodiments, a lipid composition according to the present inventioncomprises a macrolide and finds use, e.g., in immunosuppression and/orthe suppression of transplant rejection. Similarly, in some embodiments,a lipid composition according to the present invention comprises ananticancer drug as an active component, and finds use, e.g., intreatment of cancer diseases.

The methods, compositions and systems of the present invention are notlimited to use with or comprising any particular active components oragents. For example, drugs, active agents or therapeutic agents thatfind use in the methods, compositions and systems of the presentinvention include, e.g., agents that act on the peripheral nerves,adrenergic receptors, cholinergic receptors, the skeletal muscles, thecardiovascular system, smooth muscles, the blood circulatory system,synaptic sites, neuroeffector functional sites, endocrine and hormonesystems, the immunological system, the reproductive system, the skeletalsystem, the alimentary and excretory systems, the histamine system andthe central nervous system. Suitable active agents may be selected from,for example, proteins, enzymes, and hormones, nucleotides (includingsense and antisense oligonucleotides) (e.g., U.S. Pat. No. 6,126,965,2000), polynucleotide, nucleoproteins, polysaccharides, glycoproteins,lipoproteins, polypeptides, steroids. Active agents can be analgesics,anesthetics, anti-arrhythmic agents, antibiotics, antiallergic agents,antifungal agents, anticancer agents, anticoagulants, antidepressants,antidiabetic agents, anti-epilepsy agents, anti-inflammatorycorticosteroids, agents for treating Alzheimer's or Parkinson's disease,antiulcer agents, anti-protozoal agents, anxiolytics, thyroids,anti-thyroids, antiviral, anorectics, bisphosphonates, cardiac inotropicagents, cardiovascular agents, corticosteroids, diuretics, dopaminergicagents, gastrointestinal agents, hemostatics, hyper cholesterol agents,antihypertensive agents (e.g., dihydropyridines), antidepressants, andcox-2 inhibitors, immunosuppressive agents, anti-gout agents,anti-malarials, steroids, terpinoids, triterpines, retinoid, anti-ulcerH2-receptor antagonists, hypoglycemic agents, moisturizers, cosmetics,anti-migraine agents, antimuscarinic agents, anti-inflammatory agents,such as agents for treating rheumatology, arthritis, psoriasis,inflammatory bowel disease, Crohn's disease, or agents for treatingdemyelinating diseases including multiple sclerosis, ophthalmic agents,vaccines (e.g., against pneumonia, hepatitis A, hepatitis B, hepatitisC, cholera toxin B subunit, influenza virus, typhoid, plasmodiumfalciparum, diphtheria, tetanus, HSV, tuberculosis, HIV, SARS virus,perpetual pertussis, measeles, mumps and rubella vaccine (MMV),bacterial toxins, vaccinea virus, adenovirus, canary, polio virus,bacillus calmette guerin (BCG), klebsiella pneumonia, etc.), histaminereceptor antagonists, hypnotics, kidney protective agents, lipidregulating agents, muscle relaxants, neuroleptics, neurotropic agents,opioid agonists and antagonists, parasympathomimetics, proteaseinhibitors, prostaglandins, sedatives, sex hormones (e.g., estrogen,androgen), stimulants, sympathomimetics, vasodilators and xanthenes andsynthetic analogs of these species. The therapeutic agents can benephrotoxic, such as cyclosporine and amphotericin B, or cardiotoxic,such as amphotericin B and paclitaxel. Exemplary anticancer agentsinclude melphalan, chlormethine, extramustinephosphate, uramustine,ifosfamide, mannomustine, trifosfamide, streptozotocin, mitobronitol,mitoxantrone (see., e.g., international patent application WO 02/32400),methotrexate, fluorouracil, cytarabine, tegafur, idoxide, taxanes[(e.g., taxol, paclitaxel, etc., see international patent application WO00/01366; U.S. Pat. No. 5,415,869)], daunomycin or daunorubicin,epirubicin, bleomycin, etoposide, tamoxifen, hydroxytamoxifen, endoxifencarboplatin, cisplatin, paclitaxel, docetaxel, BCNU, vinca alkaloids(e.g., vincristine, vinorelbine (e.g., international patent applicationWO 03/018018, and the like) camptothecin and derivatives thereof (see,e.g., international patent publication WO 02/058622), SN 38, irinotecan(see, e.g., international patent publication WO 03/030864, and thelike), cytokines, ribozymes, interferons, oligonucleotides andfunctional anthracyclines, antibodies, cytoxines, doxorubicin, etopside,derivatives of the foregoing. Additional examples of drugs that find usein the methods, compositions and systems of the present inventioninclude, azidothymidine (AZT), acyclovir, tacrolimus, prochlorperzineedisylate, ferrous sulfate, aminocaproic acid, mecamylaminehydrochloride, procainamide hydrochloride, amphetamine sulfate,methamphetamine hydrochloride, benzamphetamine hydrochloride,isoproterenol sulfate, phenmetrazine hydrochloride, bethanecholchloride, methacholine chloride, pilocarpine hydrochloride, atropinesulfate, scopolamine bromide, isopropamide iodide, tridihexethylchloride, phenformin hydrochloride, methylphenidate hydrochloride,theophylline cholinate, cephalexin hydrochloride, diphenidol, meclizinehydrochloride, prochlorperazine maleate, phenoxybenzamine,thiethylperzine maleate, anisindone, diphenadione erythrityl tetranitrate, digoxin, isoflurophate, acetazolamide, methazolamide,bendroflumethiazide, chloropromaide, tolazamide, chlormadinone acetate,phenaglycodol, allopurinol, aluminum aspirin, methotrexate, acetylsulfisoxazole, erythromycin, hydrocortisone, hydrocorticosteroneacetate, cortisone acetate, dexamethasone and its derivatives such asbetamethasone, triamcinolone, methyl testosterone, 17-β-estradiol,ethinyl estradiol, ethinyl estradiol 3-methyl ether, prednisolone,17-α-hydroxyprogesterone acetate, 19-norprogesterone, norgestrel,norethindrone, norethisterone, norethiederone, progesterone,norgesterone, norethynodrel, aspirin, indomethacin, naproxen,fenoprofen, sulindac, indoprofen, nitroglycerin, isosorbide dinitrate,propranolol, timolol, atenolol, alprenolol, cimetidine, clonidine,imipramine, levodopa, chlorpromazine, methyldopa,dihydroxyphenylalanine, theophylline, calcium gluconate, ketoprofen,ibuprofen, cephalexin, erythromycin, haloperidol, zomepirac, ferrouslactate, vincamine, diazepam, phenoxybenzamine, diltiazem, milrinone,mandol, quanbenz, hydrochlorothiazide, ranitidine, flurbiprofen,fenufen, fluprofen, tolmetin, alclofenac, mefenamic, flufenamic,difuinal, nimodipine, nitrendipine, nisoldipine, nicardipine,felodipine, lidoflazine, tiapamil, gallopamil, amlodipine, mioflazine,lisinolpril, enalapril, enalaprilat captopril, ramipril, famotidine,nizatidine, sucralfate, etintidine, tetratolol, minoxidil,chlordiazepoxide, diazepam, amitriptyline, and imipramine. Furtherexamples are proteins and peptides which include, but are not limitedto, bone morphogenic proteins, insulin, colchicine, glucagon, thyroidstimulating hormone, parathyroid and pituitary hormones, digestivehormones, calcitonin, rennin, prolactin, corticotrophin, thyrotropichormone, follicle stimulating hormone, chorionic gonadotropin,gonadotropin releasing hormone, bovine somatotropin, porcinesomatotropin, oxytocin, vasopressin, GRF, somatostatin, lypressin,pancreozymin, luteinizing hormone, LHRH, LHRH agonists and antagonists,leuprolide, interferon's (e.g., consensus interferon, interferon α-2α,interferon α-2β, α-, β-, or γ-interferon's), interleukins, growthhormones such as human growth hormone and its derivatives such asmethione-human growth hormone and desphenylalanine human growth hormone,bovine growth hormone and porcine growth hormone, fertility inhibitorssuch as the prostaglandins, fertility promoters, growth factors such asinsulin-like growth factor, coagulation factors, pancreas hormonereleasing factor, analogues and derivatives of these compounds, andpharmaceutically acceptable salts of these compounds, or their analoguesor derivatives. The therapeutic agent can be a mixture of drugs oragents (e.g., two or more agents) that can be beneficiallyco-administered in the liposome formulation.

The inventive method is simple, rapid and less expensive method toproduce organic solvent-free aqueous liposome systems, which allow aparticularly simple and rapid inspection of foreign particles.Furthermore, the liposome system produced according to the inventivemethod shows highly reproducible particle sizes, with average particlesize below 5 micron, preferably between 50 nm and 1 micron. It is alsopossible to filter the product through sterile filtration known in theart. The duration of the extrusion, or the high pressure splithomogenization is chosen to be sufficiently long for the liposomes toshow the desired average diameter. Said extrusion, high pressure splithomogenization is performed until liposomes possess a mean diameterbetween 50 nm and 1 micron.

The liposome system produced according to the present inventive methodcan be filled directly in corresponding ampoules in a condition ready touse, and lyophilize the product after the adding the desired amount ofcarbohydrate known in the art, whereby lyophilization constitute thebest method of water drying. This gives liposome system in powder form,which can be re-constituted into the vesicles by the addition ofsuitable amount of water for injection, normal saline or 5% dextrosewith gentle shaking. It is not necessary to subject the liposome systemformed after the addition of injectable water to extensive agitation orhigh pressure split homogenization.

The methods and compositions of the present invention are used to treata disease caused by fungal or bacterial infection. In some embodiments,the methods and compositions of the present invention are used to treata fungal disease caused by at least one of the fungus selected from thegroup of fungus consisting of Acremonium sp., Aspergillus fumigatus,Aspergillus pneumonia, Blastomyces dermatitidis, candida albicans,Candida guillermondi, Candida tropicalis, Coccidioides immitis,Cryptococcus neoformans, Fusarium sp., Histoplasma capsulatum, Mucormucedo, Rhodotorula sp., Sporothrix schenckii, Acanthamoeba polyphaga,Entomophthora sp., Histoplasma capsulatumm Leishmania brasiliensis,Rhizopus sp., Rhodotorula sp., Torulopsis glabrata, Paracoccidioidesbrasiliensis. Additional fungal pathogens include Trichosporon, Muco,Alternaria, Bipolaris, Curvularia, etc.

In some embodiments, the methods and compositions of the presentinvention are used to treat disease caused by a species of Leishmania,for example, in some embodiments, the methods and compositions of thepresent invention are used to treat Visceral Leishmaniasis.

In some embodiments, the methods and compositions of the presentinvention are used to treat a viral infection, e.g. a viral infectioncaused by human immunodeficiency virus (HIV), herpes simplex viruses(HSV-1 and HSV2), hepatitis C virus (HCV) or cytomegalovirus (CMV).

In some embodiments, the present inventions comprise a method oftreating a cell with amphotericin B with or without deoxycholate,preparing a composition according as described herein, and exposing thecells to the composition. In some preferred embodiments, the exposing ofthe cell occurs in vivo, e.g., in a patient or subject.

It is contemplated that in some embodiments, the exposing of a cell in asubject comprises oral delivery of the composition to the subject, whilein other embodiments; the exposing of a cell comprises intravenousdelivery of the composition to the subject. Routes of delivery of thecomposition to the subject that find use in the present inventioninclude but are not limited to subcutaneous delivery, parenteraldelivery, intraperitoneal delivery, rectal delivery, vaginal deliveryand/or topical delivery. In some preferred embodiments, the subject is amammal. In some particularly preferred embodiments, the mammal is human.

DEFINITIONS

The term “lipid composition” as used herein refers to amphotericcompounds which are capable of liposome formation, vesicle formation,micelle formation, emulsion formation, and are substantially non-toxicwhen administered. The lipid composition may include without limitationegg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), soyphosphatidylcholine (SPC), hydrogenated soy phosphatidylcholine (HSPC),dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol(DMPG), Dipalmitoylphosohatidylcholine (DPPC),disteroylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol(DMPG), cholesterol (Chol), cholesterol sulfate and its salts (CS),cholesterol hemisuccinate and its salts (Chems), cholesterol phosphateand its salts (CP), cholesterylphospholine and other hydroxycholesterolor amino cholesterol derivatives.

As used herein, the term “aqueous” as used in reference to a solvent,fluid, or system, refers to a water-based solvent, fluid or system thatdoes not contain any organic solvents.

As used herein, the term “aqueous system” as used in reference toproduction of a complex comprising at least one active compound and atleast one lipid refers to a process or method of production, or to theset of materials used in such production, that contain or comprise useof water-based solvents and lipids but do not contain or comprise use oforganic solvents.

As used herein, the term “organic solvent” refers to a carbon-containingchemical, generally in liquid form, used to dissolve another substance.Examples of organic solvents include but are not limited to alcohols,glycols, ethers, dimethoxyethane, acetone, chloroform, dimethylsulfoxide, hexane, toluene, tetrahydrofuron (THF), methylene chlorideand the like.

The term “encapsulating amount” refers to the amount of lipid necessaryto encapsulate the poorly soluble compound and form liposome or lipidicparticles of appropriate mean particle size less than 5,000 nm indiameter, preferably between 30-1000 nm. The encapsulating amount willdepend on the pharmaceutically active compounds and process conditionsselected, but in general range in between from 2:1 to about 1:100compound:lipid ratio; preferably about 1:1 to about 1:50.

The term “lipidic particle” as used herein refers to particles ofundefined structure which consist of a suitable lipid and anencapsulated or complexed pharmaceutically active compound. Polyeneantibiotics at high antibiotic:lipid ratios typically form lipidicparticles rather than liposomes, due to the polyene structure and itsinteraction with the lipid. Lipidic particles may have a lamellarstructure but are not required to exhibit any defined structure.

As used herein, the term “effective amount” refers to the amount of anactive composition (e.g., a pharmaceutical compound or compositionprovided as a component in a lipid formulation) sufficient to effectbeneficial or desired results. An effective amount can be administeredin one or more administrations, applications or dosages and is notintended to be limited to a particular formulation or administrationroute.

As used herein, the terms “active” or “pharmaceutically active” as usedin reference to an agent, composition, or compound, refers to an agentthat, upon administration or application, causes a beneficial, desired,or expected result. The administration may be in one or moreadministrations, applications or dosages and is not intended to belimited to a particular formulation or administration route. The term isnot limited to any particular level of activity. For example, a lipidformulation of an active agent need not have the same level of activityas a different formulation of an active agent, so long as the activeagent in the lipid formulation is sufficiently active that an effectiveamount of the active agent can be administered by administration of thelipid formulation of the agent.

The terms “agent” and “compound” are used herein interchangeably torefer to any atom, molecule, mixture, or more complex composition havingan attributed feature. For example, an “active agent” or “activecompound” refers to any atom, molecule, preparation, mixture, etc.,that, upon administration or application, causes a beneficial, desired,or expected result.

As used herein, the term “administration” refers to the act of giving adrug, prodrug, or other active agent, or therapeutic treatment (e.g.,compositions of the present invention) to a physiological system (e.g.,a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs).Exemplary routes of administration to the human body can be through theeyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs(inhalant), rectal, vaginal, oral mucosa (buccal), ear, by injection(e.g., intravenously, subcutaneously, intratumorally, intraperitoneally,etc.) and the like. Administration may be in one or moreadministrations, applications or dosages, and is not intended to belimited to a particular administration route.

As used herein, the term “co-administration” refers to theadministration of at least two agent(s) (e.g., two separate lipidcompositions, containing different active compounds) or therapies to asubject. In some embodiments, the co-administration of two or moreagents or therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy. Those ofskill in the art understand that the formulations and/or routes ofadministration of the various agents or therapies used may vary. Theappropriate dosage for co-administration can be readily determined byone skilled in the art. In some embodiments, when agents or therapiesare co-administered, the respective agents or therapies are administeredat lower dosages than appropriate for their administration alone. Thus,co-administration is especially desirable in embodiments where theco-administration of the agents or therapies lowers the requisite dosageof a potentially harmful (e.g., toxic) agent(s).

As used herein, the term “toxic” refers to any detrimental or harmfuleffects on a subject, a cell, or a tissue as compared to the same cellor tissue prior to the administration of the toxicant.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent (e.g., an active pharmaceutical compound)with a carrier, inert or active (e.g., a phospholipid), making thecomposition especially suitable for diagnostic or therapeutic use invitro, in vivo or ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, the term “topically” refers to application of thecompositions of the present invention to the surface of the skin andmucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory,or nasal mucosa, and other tissues and cells which line hollow organs orbody cavities).

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, emulsions (e.g., such asan oil/water or water/oil emulsions), and various types of wettingagents, any and all solvents, dispersion media, coatings, sodium laurylsulfate, isotonic and absorption delaying agents, disintrigrants (e.g.,potato starch or sodium starch glycolate), and the like. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers, and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. (1975), incorporated herein by reference). Moreover, in certainembodiments, the compositions of the present invention may be formulatedfor horticultural or agricultural use. Such formulations include dips,sprays, seed dressings, stem injections, sprays, and mists.

As used herein, the term “pharmaceutically acceptable salt” refers toany salt (e.g., obtained by reaction with an acid or a base) of acompound of the present invention that is physiologically tolerated inthe target subject (e.g., a mammalian subject, and/or in vivo or exvivo, cells, tissues, or organs). “Salts” of the compounds of thepresent invention may be derived from inorganic or organic acids andbases. Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g.,sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide,iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate,persulfate, phenylpropionate, picrate, pivalate, propionate, succinate,tartrate, thiocyanate, tosylate, undecanoate, and the like. Otherexamples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like. For therapeutic use,salts of the compounds of the present invention are contemplated asbeing pharmaceutically acceptable. However, salts of acids and basesthat are non-pharmaceutically acceptable may also find use, for example,in the preparation or purification of a pharmaceutically acceptablecompound.

For therapeutic use, salts of the compounds of the present invention arecontemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable compound.

The term “Polyethylene glycol (PEG)” includes polymers of lower alkyleneoxide, in particular ethylene oxide (polyethylene glycols) having anesterifiable hydroxyl group at least at one end of the polymer molecule,as well as derivatives of such polymers having esterifiable carboxygroups. Polyethylene glycols of an average molecular weight ranging from200-20,000 are preferred; those having an average molecular weightranging from 500-2000 are particularly preferred.

The use of terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising”, “including”, “having”, and “containing”are to be construed as open-ended terms (i.e. meaning “including but notlimited to”) unless otherwise noted. The use of any and all examples, orexemplary language (e.g., “such as”) provided herein, is intended merelyto better illuminate the invention and does not pose a limitation on thescope of the invention unless otherwise claimed. No language in thespecifications should be construed as indicating any non-claimed elementas essential to the practice of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the preparation of suspension, liposomes,lipid complex, or micelles in an aqueous system. The inventivepreparation comprises at least one phospholipid, such as Soyaphosphatidylcholine, in aqueous media with therapeutically activeinsoluble or poorly soluble compound.

Particular embodiments of the invention are described in the Summary,and in this Detailed Description of the Invention. Although theinvention has been described in connection with specific embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. For example, the compositions andmethods of the present invention are described in connection withparticular polyene antibiotics, such as amphotericin B with or withoutdeoxycholate. It should be understood that the present invention is notlimited to methods or compositions using or comprising amphotericin B.In particular, the present invention relates to composition and methodof preparing organic solvent-free formulation comprising one or moreactive compounds.

The present invention also relates to compositions and methods ofdelivering anticancer drugs, for example, docetaxel and paclitaxel, andimmunosuppressant agents, such as tacrolimus and sacrolimus.

The present invention relates to compositions and methods for deliveringpolyene antibiotics that reduce the toxicity of the antibiotic to thehost being treated. Several formulation strategies have been used toreduce the nephrotoxicity of amphotericin B. For example, certain lipidbased formulations of amphotericin B have been found to reduce toxicityand to increase tolerance and therapeutic efficacy (Janoff, A. et al.U.S. Pat. No. 6,406,713, 2002, which is incorporated herein by referencein its entirety).

Amphotericin B is insoluble in aqueous solution and before it can beused clinically as an antifungal agent, a vehicle (carrier) has to beadded to form dispersion. The commercial preparation of amphotericin B,FUNGIZONE is a mixture of amphotericin B, a detergent deoxycholate, anda buffer. When suspended in a glucose solution, FUNGIZONE formscolloidal dispersion suitable for intravenous injection. (Brajtburg, J.et al. 1990). FUNGIZONE the first marketed formulation of amphotericin Bwith deoxycholate remains the gold standard in spite of its renaltoxicity. FUNGIZONE is currently marketed as lyophilized cake providing50 mg amphotericin B and 41 mg of deoxycholate with 20.2 mg of sodiumphosphates as a buffer.

In an effort to improve the delivery of amphotericin B in the treatmentof fungal diseases, several liposome formulations have been designed.Liposomal composition containing egg phosphatidylcholine, dipalmitoylphosphatidylethanolamine, and cholesterol in molar ratio of 6:1:3 weremore efficient in improving the therapeutic index as compared to freedrug. Further, amphotericin B intercalated into mannosylated liposomesis less toxic and more effective as fungal killer (Ahmad, I. et al.,1989, 1990, 1991).

AMBISOME is a lyophilized formulation of amphotericin B incorporatedinto unilamellar liposomes formed from soy phosphatidylcholine,distearoylphosphatidylglycerol, and cholesterol. AMBISOME binds to thefungal cells, resulting in death of the fungus. (Adler-Moore, Jill P. etal., 1994; Adler-Moore et al. 1993). AMBISOME formulation has greatlyreduced the toxicity of amphotericin B, and high plasma concentrationsand tissue accumulations of drug can be achieved with non-toxic doses ofAMBISOME (Proffitt et al, U.S. Pat. No. 5,965,156, 1999; Proffitt, R. T.1991).

ABELCET is liposome formulation consists of a 1:1 ratio of amphotericinB in combination with a 7:3 ratio of dimyristoyl phosphatidylcholine todimyristoyl phosphatidylglycerol. The resulting complex forms a tightlypacked ribbon structure, approximately 250 nm diameter. The safety andefficacy of ABELCET have been extensively evaluated in clinical studiesand have shown that ABELCET is, in general less toxic than amphotericinB deoxycholate (Lister, J. 1996; Walsh, T. J. et al 1997).

In order to reduce the toxicity of amphotericin B, a new formulation hasbeen developed consisting of a cholesteryl sulfate complex withamphotericin B, the amphotericin B colloidal dispersion (AMPHOTEC).AMPHOTEC is a stable complex of amphotericin B and cholesteryl sulfatein a 1:1 molar ratio. In vitro studies with fresh human blood have shownthat the drug-lipid complex does not result in hemolysis of erythrocytesand that binding to plasma lipoproteins is less than that observed withFUNGIZONE However, the pharmacokinetics of amphotericin B followinginfusions of ABCD does not differ significantly from those of FUNGIZONE.(Szoka, F. C. Jr. U.S. Pat. No. 5,277,914 A 1994; Abra, R. and Guo, L.S. U.S. Pat. No. 5,194,266, 1993; Abra, R. U.S. Pat. No. 5,032,582,1991; Abra, R. U.S. Pat. No. 4,822,777, 1989; Abra, R. et al. PCT ApplWO8701933, 1987; Sanders, S. et al. 1991).

The various lipid formulations of amphotericin B described above,however, are still capable of producing all of the toxicities associatedwith amphotericin B alone, although nephrotoxicity is reduced to someextent with all these formulations.

The present invention provides formulations using new lipid compositionsthat reduce the toxicities associated with active compounds such asamphotericin B deoxycholate.

The present invention provides compositions and methods for deliveringactive compounds such as polyene antibiotics, e.g., to a mammalian host.Examples of polyene antibiotics that find use in the present inventioninclude but are not limited to amphotericin B deoxycholate (FUNGIZONE),Nystatin (Nys), Natamycin, Candicidin, Aureofungin A, Aureofungin B,Hamycin A, Hamycin B, Trienin, Pimaricin, Etruscomycin, Chainin,Dermostatin, Filipin, and Lymphosarcin. In some preferred embodiments,the present invention comprises compositions and methods for thedelivery of amphotericin B deoxycholate (FUNGIZONE) to a mammalian host.Any suitable amount of an active compound, e.g., polyene antibioticssuch as amphotericin B deoxycholate, can be used. Suitable amounts ofpolyene antibiotic are those amounts that can be stably incorporatedinto the complexes of the present invention.

The present invention provides compositions and methods of deliveringanticancer drugs, e.g., to a mammalian host. Examples of anticancerdrugs that find use in the present invention include but are not limitedto paclitaxel, docetaxel, doxorubicin, daunomycin, epirubicin,etoposide, tamoxifen, endoxifen, vincristine anthracycline, and thelike. Any suitable amount of anticancer drugs can be used. Suitableamounts of anticancer drugs are those amounts that can be stablyincorporated into the complexes of the present invention.

The present invention provides compositions and method of deliveringimmunosuppressant agents. Examples of immunosuppressant agents that finduse in the present invention include but not limited to tacrolimus andsacrolimus. Any suitable amount of immunosuppressant agents can be used.Suitable amounts of immunosuppressant agents are those amounts that canbe incorporated into the complexes of the present invention.

The present inventions provide compositions and method for treatingrejection reactions caused by the transplantations organs and tissues.Examples of organs and tissue transplantation include but not limited toheart, kidney, liver, lung, bone marrow, skin, cornea, pancreas, smallintestine, muscle, limb, myoblast, intervertebral disc, cartilage, bone,blood vessel, nervous system, esophagus and the like.

In some embodiments, the present invention comprises a lipid complexwith active compound (for example, amphotericin B with or withoutdeoxycholate) in which the complex contains lipid or a mixture oflipids. In some embodiments, the complexes are in the form of micelles,emulsions or mixture of micelles and vesicles. The micelles of thepresent invention can be in the form, e.g., of monomeric, dimeric,polymeric or mixed micelles. In some embodiments, the complexesincluding micelles, emulsions or mixture of micelles and vesicles arepredominately in the size range of 50 nm-20 micron, while in somepreferred embodiments, the micelles and emulsions are in the size rangeof 50 nm-5 micron. In the complexes of the present invention, theantibiotic can be bound to the lipid by covalent, hydrophobic,electrostatic, hydrogen, or other bonds, and is considered “bound” evenwhere the antibiotic is simply entrapped within the interior of lipid.

In some embodiments, active agent-lipid complexes (for example,amphotericin B-lipid complexes with or without deoxycholate) containcholesterol or cholesterol derivatives. Examples of cholesterolderivatives that find use in the present invention include but are notlimited to cholesteryl sulfate, cholesteryl hemisuccinate, cholesterylsuccinate, cholesteryl oleate, cholesteryl linoleate, cholesteryleicosapentenoate, cholesteryl linolenate, cholesteryl arachidonate,cholesteryl palmitate, cholesteryl stearate, cholesteryl myristate,polyethylene glycol derivatives of cholesterol (cholesterol-PEG), watersoluble cholesterol (for example, cholesterol methyl-β-cyclodextrin),coprostanol, cholestanol, or cholestane, cholic acid, cortisol,corticosterone or hydrocortisone and 7-dehydrocholesterol. In somepreferred embodiments, the cholesterol or cholesterol derivatives arecomplexed with an active compound at low pH (e.g., in the range of aboutpH 1.0 to pH 4.0).

In some preferred embodiments, the compositions also include α-, β-,γ-tocopherols, vitamin E, calciferol, organic acid derivatives of α-,β-, γ-tocopherols, such as α-tocopherol hemisuccinate (THS),α-tocopherol succinate, or mixtures thereof.

In some preferred embodiments, active agent-lipid complexes (forexample, amphotericin B-lipid complexes, with or without deoxycholate)contain sterols. Examples of sterols that find use in the presentinvention include β-sitosterol, stigmasterol, stigmastanol, lanosterol,α-spinasterol, lathosterol, campesterol and/or mixtures thereof.

Compositions of the present invention also include active compounds (forexample, amphotericin B complexes with or without deoxycholate) withfree and/or salts or esters of fatty acid. In some preferredembodiments, fatty acids range from carbon chain lengths of about C₂ toC₃₄, preferably between about C₄ and about C₂₄, and include tetranoicacid (C_(4:0)), pentanoic acid (C_(5:0)), hexanoic acid (C_(6:0)),heptanoic acid (C_(7:0)), octanoic acid (C_(8:0)), nonanoic acid(C_(9:0)), decanoic acid (C_(10:0)), undecanoic acid (C_(11:0)),dodecanoic acid (C_(12:0)), tridecanoic acid (C_(13:0)), tetradecanoic(myristic) acid (C_(14:0)), pentadecanoic acid (C_(15:0)), hexadecanoic(palmatic) acid (C_(16:0)), heptadecanoic acid (C_(17:0)), octadecanoic(stearic) acid (C_(18:0)), nonadecanoic acid (C_(19:0)), eicosanoic(arachidic) acid (C_(20:0)), heneicosanoic acid (C_(21:0)), docosanoic(behenic) acid (C_(22:0)), tricosanoic acid (C_(23:0)), tetracosanoicacid (C_(24:0)), 10-undecenoic acid (C_(11:1)), 11-dodecenoic acid(C_(12:1)), 12-tridecenoic acid (C_(13:1)), myristoleic acid (C_(14:1)),10-pentadecenoic acid (C_(15:1)), palmitoleic acid (C_(16:1)), oleicacid (C_(18:1)), linoleic acid (C_(18:2)), linolenic acid (C_(18:3)),eicosenoic acid (C_(20:1)), eicosdienoic acid (C₂₀₂), eicosatrienoicacid (C₂₀₃), arachidonic acid (cis-5,8,11,14-eicosatetraenoic acid), andcis-5,8,11,14,17-eicosapentaenoic acid, among others. Other fatty acidchains also can be employed in the compositions. Examples of suchinclude saturated fatty acids such as ethanoic (or acetic) acid,propanoic (or propionic) acid, butanoic (or butyric) acid, hexacosanoic(or cerotic) acid, octacosanoic (or montanic) acid, triacontanoic (ormelissic) acid, dotriacontanoic (or lacceroic) acid, tetratriacontanoic(or gheddic) acid, pentatriacontanoic (or ceroplastic) acid, and thelike; monoethenoic unsaturated fatty acids such as trans-2-butenoic (orcrotonic) acid, cis-2-butenoic (or isocrotonoic) acid, 2-hexenoic (orisohydrosorbic) acid, 4-decanoic (or obtusilic) acid, 9-decanoic (orcaproleic) acid, 4-dodecenoic (or linderic) acid, 5-dodecenoic (ordenticetic) acid, 9-dodecenoic (or lauroleic) acid, 4-tetradecenoic (ortsuzuic) acid, 5-tetradecenoic (or physeteric) acid, 6-octadecenoic (orpetroselenic) acid, trans-9-octadecenoic (or elaidic) acid,trans-11-octadecenoic (or vaccinic) acid, 9-eicosenoic (or gadoleic)acid, 11-eicosenoic (or gondoic) acid, 11-docosenoic (or cetoleic) acid,13-decosenoic (or erucic) acid, 15-tetracosenoic (or nervonic) acid,17-hexacosenoic (or ximenic) acid, 21-triacontenoic (or lumequeic) acid,and the like; dienoic unsaturated fatty acids such as 2,4-pentadienoic(or β-vinylacrylic) acid, 2,4-hexadienoic (or sorbic) acid,2,4-decadienoic (or stillingic) acid, 2,4-dodecadienoic acid,9,12-hexadecadienoic acid, cis-9, cis-12-octadecadienoic (or α-linoleic)acid, trans-9, trans-12-octadecadienoic (or linlolelaidic) acid,trans-10,trans-12-octadecadienoic acid, 11,14-eicosadienoic acid,13,16-docosadienoic acid, 17,20-hexacosadienoic acid and the like;trienoic unsaturated fatty acids such as 6,10,14-hexadecatrienoic (orhiragonic) acid, 7,10,13-hexadecatrienoic acid, cis-6,cis-9-cis-12-octadecatrienoic (or γ-linoleic) acid, trans-8,trans-10-trans-12-octadecatrienoic (or β-calendic) acid, cis-8,trans-10-cis-12-octadecatrienoic acid, cis-9,cis-12-cis-15-octadecatrienoic (or α-linolenic) acid, trans-9,trans-12-trans-15-octadecatrienoic (or α-linolenelaidic) acid, cis-9,trans-11-trans-13-octadecatrienoic (or α-eleostearic) acid, trans-9,trans-11-trans-13-octadecatrienoic (or β-eleostearic) acid, cis-9,trans-11-cis-13-octadecatrienoic (or punicic) acid,5,8,11-eicosatrienoic acid, 8,11,14-eicosatrienoic acid and the like;tetraenoic unsaturated fatty acids such as 4,8,11,14-hexadecatetraenoicacid, 6,9,12,15-hexadecatetraenoic acid, 4,8,12,15-octadecatetraenoic(or moroctic) acid, 6,9,12,15-octadecatetraenoic acid,9,11,13,15-octadecatetraenoic (or α- or β-parinaric) acid,9,12,15,18-octadecatetraenoic acid, 4,8,12,16-eicosatetraenoic acid,6,10,14,18-eicosatetraenoic acid, 4,7,10,13-docasatetraenoic acid,7,10,13,16-docosatetraenoic acid, 8,12,16,19-docosatetraenoic acid andthe like; penta- and hexa-enoic unsaturated fatty acids such as4,8,12,15,18-eicosapentaenoic (or timnodonic) acid,4,7,10,13,16-docosapentaenoic acid, 4,8,12,15,19-docosapentaenoic (orclupanodonic) acid, 7,10,13,16,19-docosapentaenoic, 4,7,10,13,16,19-docosahexaenoic acid, 4,8,12,15,18,21-tetracosahexaenoic (ornisinic) acid and the like; branched-chain fatty acids such as3-methylbutanoic (or isovaleric) acid, 8-methyldodecanoic acid,10-methylundecanoic (or isolauric) acid, 11-methyldodecanoic (orisoundecylic) acid, 12-methyltridecanoic (or isomyristic) acid,13-methyltetradecanoic (or isopentadecylic) acid, 14-methylpentadecanoic(or isopalmitic) acid, 15-methylhexadecanoic, 10-methylheptadecanoicacid, 16-methylheptadecanoic (or isostearic) acid, 18-methylnonadecanoic(or isoarachidic) acid, 20-methylheneicosanoic (or isobehenic) acid,22-methyltricosanoic (or isolignoceric) acid, 24-methylpentacosanoic (orisocerotic) acid, 26-methylheptacosanoic (or isomonatonic) acid,2,4,6-trimethyloctacosanoic (or mycoceranic or mycoserosic) acid,2-methyl-cis-2-butenoic(angelic)acid, 2-methyl-trans-2-butenoic (ortiglic) acid, 4-methyl-3-pentenoic (or pyroterebic) acid and the like.

In certain preferred embodiments, active compounds (for example,amphotericin B-lipid complexes with or without deoxycholate) comprisephospholipids. Any suitable phospholipids can be used. For example,phospholipids can be obtained from natural sources or chemicallysynthesized. Examples of phospholipids that find use in the presentinvention include phosphatidylethanolamine (PE), phosphatidylglycerol(PG), phosphatidylserine (PS), phosphatidylcholine (PC),phosphatidylinositol (PI), phosphatidic acid (PA), sphingomyelin and thelike, either used separately or in combination. Phosphatidylglycerolsmay be having short chain or long chain, saturated or unsaturated suchas dimyristoylphosphatidylglycerol, dioleoylphosphatidylglycerol,distearoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol,diarachidonoylphosphatidylglycerol, short chain phosphatidylglycerol(C₆-C₈), and mixtures thereof. Examples of phosphatidylcholines includesdimyristoylphophatidylcholine, distearoylphosphatidylcholine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,diarachidonoylphosphatidylcholine, egg phosphatidylcholine, soyphosphatidylcholine or hydrogenated soy phosphatidylcholine can be used,as can mixtures thereof.

In some embodiments, the present invention provides compositionscomprising at least one active compound (for example, amphotericin Bwith or without deoxycholate) and derivatives of mono-, di- andtri-glycerides. Examples of the glycerides that find use in the presentinvention include but are not limited to 1-oleoyl-glycerol (monoolein)and 1, 2-dioctanoyl-sn-glycerol.

Another aspect of the invention is to complex at least one activecompound (for example, amphotericin B with or without deoxycholate) withat least one functionalized phospholipid, including but not limited tophosphatidylethanolamine, phosphatidylthioethanol,N-biotinylphosphatidylethanolamine, and phosphatidylethylene glycol. Insome preferred embodiments, amphotericin B with or without deoxycholateis complexed with dioleoylphosphatidylethanolamine.

Another aspect of the invention is to complex at least one activecompound (for example, amphotericin B with or without deoxycholate) withat least one carbohydrate-based lipid. Examples of carbohydrate-basedlipids that find use in the present invention include but are notlimited to galactolipids, mannolipids, galactolecithin and the like.

Yet another aspect of the invention is to complex at least one activecompound (for example, amphotericin B with or without deoxycholate) withderivatives of phospholipids such as pegylated phospholipids. Examplesinclude but not limited to the polyethylene glycol (Pegylated, PEG)derivatives of distearoylphosphatidylglycerol,dimyristoylphosphatidylglycerol, dioleoylphosphatidylglycerol and thelike.

Another further aspect of the present invention provides compositionscomprising at least one active compound (for example, amphotericin Bwith or without deoxycholate) and polyethylene glycol (PEG) and one ormore lipids.

According to another aspect, the present invention provides compositionscomprising at least one active compound (for example, amphotericin Bwith or without deoxycholate) complexed with one or more lipids. Exampleincludes compositions comprising amphotericin B with or withoutdeoxycholate, cholesterol or cholesterol derivatives and one or morephospholipids. Other examples of compositions according to the inventioninclude amphotericin B with or without deoxycholate, β-sitosterol, andone or more phospholipids. In some preferred embodiments, thecomposition of the present invention comprises amphotericin B, with orwithout deoxycholate, cholesteryl sulfate and hydrogenated soyphosphatidylcholine or soy phosphatidylcholine.

The composition of the present invention can be made by dissolving anactive compound, for example, amphotericin B deoxycholate (e.g.,Fungizone®) in water at a concentration of about 0.5 mg/mL to about 25mg/mL. In some embodiments, the antibiotic is dissolved at aconcentration between 1 mg/mL and about 20 mg/mL. In certain preferredembodiments, the antibiotic is dissolved at a concentration of between 1mg/mL and 10 mg/mL. In particularly preferred embodiments, theantibiotic is dissolved at a concentration of between 1 mg/mL and 5mg/mL.

In some embodiments, compositions of the present invention contain about2.5% to about 95% by weight of total lipid, preferably about 10% toabout 90% by weight of total lipid or more, preferably about 20% toabout 90% by weight of total lipid.

In some embodiments, compositions of the present invention contain atleast one active compound (for example, amphotericin B, with or withoutsodium deoxycholate) and lipid(s) in mole ratio between 1:1 to 1:100,e.g., in between 1:1 and 1:20 molar ratio or in between 1:1 and 1:30molar ratio or in between 1:1 and 1:40 molar ratio or in between 1:1 and1:50 molar ratio, in between 1:1 and 1:60 molar ratio, in between 1:1and 1:70 molar ratios, and in between 1:1 and 1:80 molar ratios. As usedherein, the term “in between” is inclusive of the limits of a recitedrange. For example, a mole ratio “in between” 1:1 and 1:20 molar ratioincludes ratios of 1:1 and 1:20.

In certain preferred embodiments, compositions of the present inventioncontain at least one active compound (for example, amphotericin B, withor without sodium deoxycholate), cholesteryl sulfate and hydrogenatedsoy phosphatidylcholine. Such compositions include amphotericin B andsodium deoxycholate in mole ratio of 1:2.

In certain preferred embodiments, the mole ratio of active compound (forexample, amphotericin B) and cholesteryl sulfate in a compositioncontaining active compound (for example, amphotericin B), sodiumdeoxycholate, cholesteryl sulfate and hydrogenated soyphosphatidylcholine is in between 1:1 and 1:20, such as in between 1:1and 1:10, or in between 1:1 and 1:5 or 1:1 and 1:2. In particularlypreferred embodiments, the mole ratio of active compound (for example,amphotericin B) and cholesteryl sulfate is in between 1:1 and 1:5.

In certain preferred embodiments, the mole ratio of active compound (forexample, amphotericin B) and hydrogenated soy phosphatidylcholine in acomposition containing active compound (for example, amphotericin B,with or without sodium deoxycholate), cholesteryl sulfate andhydrogenated soy phosphatidylcholine is in between about 1:1 and 1:90,e.g., in between 1:1 and 1:70 or 1:1 and 1:60 or 1:1 and 1:50 or 1:1 and1:40 and 1:1 and 1:30. In particularly preferred embodiments, the moleratio of active compound (for example, amphotericin B) and hydrogenatedsoy phosphatidylcholine is in between 1:5 and 1:60.

In certain preferred embodiments, the mole ratio of active compound (forexample, amphotericin B) and soy phosphatidylcholine in a compositioncontaining active compound (for example, amphotericin B), with orwithout sodium deoxycholate, cholesteryl sulfate and soyphosphatidylcholine is in between 1:1 and 1:90, e.g., in between 1:1 and1:70 or 1:1 and 1:60 or 1:1 and 1:50 or 1:1 and 1:40 and 1:1 and 1:30.In particularly preferred embodiments, the mole ratio of active compound(for example, amphotericin B) and soy phosphatidylcholine is in between1:5 and 1:60.

In some embodiments, compositions of the present invention containactive compound (for example, amphotericin B) and total lipids havingweight-to-weight ratio between 1:1 to 1:100 ratio such as in between 1:1and 1:20 ratio or in between 1:1 and 1:30 ratio or in between 1:1 and1:40 ratio or in between 1:1 and 1:50 ratio, or in between 1:1 and 1:60ratio, or in between 1:1 and 1:70 ratio, and in between 1:1 and 1:80ratio, or in between 1:1 and 1:90 ratio.

In some embodiments, the mole ratio of cholesterol or cholesterylderivative (such as cholesteryl sulfate) and one or more phospholipids(for example, soy phosphatidylcholine) is in between 1:1 and 1:90, e.g.,in between 1:1 and 1:70 or 1:1 and 1:60 or 1:1 and 1:50 or 1:1 and 1:40and 1:1 and 1:30. In particularly preferred embodiments, the mole ratioof cholesterol derivative (for example, cholesteryl sulfate) and soyphosphatidylcholine is in between 1:1 and 1:20.

In some embodiments, the methods of the present invention involvedissolving active compound, e.g., amphotericin B (with or withoutdeoxycholate), in water and mixing the dissolved antibiotic and thelipid(s) together. The active compound-lipid complex solution can befiltered through suitable filters to control the size distribution ofthe formed complexes.

In some embodiments, the method of the present invention involves mixinglipid(s) and sodium deoxycholate together in water and then addingactive compound (for example, amphotericin B). The active compound-lipidcomplex solution can be filtered through suitable filters to control thesize distribution of the formed complexes.

In some embodiments, the method comprises mixing amphotericin B andcholesteryl derivative, for example cholesteryl sulfate in water orbuffer having pH in the range of 1 to 3.0 and can be heated if desiredat temperature ranging from 25° C. to 60° C. The resulting suspension isthen mixed with phospholipids, for example soy phoaphatidylcholine orhydrogenated soy phosphatidylcholine in water or buffer and the pH isadjusted with suitable base or buffer so the resulting suspensionattains a pH ranging between 5.00 and 8.00. The acidic pH can beachieved by any suitable acid such as hydrochloric acid, phosphoric acidand the like. Examples of base or buffer includes but not limited tosodium succinate dibasic, sodium acetate, sodium phosphate monobasic,sodium phosphate dibasic, sodium phosphate tribasic, sodium hydroxide,and the like. The composition may further contain sugar. Examples ofsugars includes but not limited to sucrose, lactose, dextrose, trehalosemaltose, and the like. The percentage of sugar may range from 5% toabout 25%. The resulting suspension can be homogenized or sonicated toreduce the particle size. In some embodiments, the hydrated suspensionis filtered through suitable filters to control the size distribution ofthe formed complexes. In some embodiments, the hydrated composition canbe lyophilized to obtain the composition in powder form. In someembodiments, the hydrated composition can be autoclaved.

In some embodiments, the present invention comprises mixing amphotericinB, sodium deoxycholate, and one or more lipids in any suitable sequencesuch that the resulting composition of the present invention comprisesamphotericin B, sodium deoxycholate and one or more lipids. For example,in some embodiments, the method comprises of mixing amphotericin B in asolution containing sodium deoxycholate in water and then adjusting thepH with sodium hydroxide until the amphotericin B is completelydissolved. Lipids such as soy phosphatidylcholine are then added to theamphotericin B-sodium deoxycholate solution, followed by one more lipid,such as cholesteryl sulfate. The amphotericin B-lipid complex solutioncan be filtered through suitable filters to control the sizedistribution of the formed complexes.

In some embodiments, the present invention comprises mixing activecompound (for example, amphotericin B), and one or more lipids in anysuitable sequence such that the resulting composition of the presentinvention comprises active compound (for example, amphotericin B), andone or more lipids. For example, in some embodiments, the methodcomprises of mixing amphotericin B in water and then adjusting the pHwith sodium hydroxide until the amphotericin B is completely dissolved.Lipids such as soy phosphatidylcholine are then added to theamphotericin B solution, followed by one more lipid, such as cholesterylsulfate. The amphotericin B-lipid complex solution can be filteredthrough suitable filters to control the size distribution of the formedcomplexes. In another embodiment the amphotericin B and cholesterylsulfate is mixed at any desired pH such as at low pH for example pH inbetween 1.00 and 4.00 or at higher pH for example, pH in between 9.00and 12.00. The pH is then adjusted with suitable base or buffer toattain the pH of the resulting suspension in the range between 4.00 to8.00 and then mixed with phospholipids, for example soyphosphatidylcholine or hydrogenated phosphatidylcholine.

In some embodiments, the method of preparation of the present inventioncomprises heating a composition comprising active compound (for example,amphotericin B in water) with or without deoxycholate and one or morelipids. In some embodiments, heating is at temperatures ranging from30-121° C. In some preferred embodiments, heating is at a temperaturebetween 40-80° C., while in some particularly preferred embodiments,heating is at a temperature between 40-70° C. In some embodiments, thehydrated composition can be autoclaved.

In some embodiments, the method of preparation of present inventioncomprising mixing active compound (for example, Tacrolimus), cholesterylderivative (for example, cholesteryl sulfate) and phosphatidylcholinesuch as soy phosphatidylcholine or hydrogenated soy phosphatidylcholinein water or buffer. The resulting suspension can be homogenized orsonicated at any desired temperature ranging from 20-60° C. Examples ofbase or buffer includes but not limited to sodium succinate dibasic,sodium acetate, sodium phosphate monobasic, sodium phosphate dibasic,sodium phosphate tribasic, sodium hydroxide, and the like. Thecomposition may further contain sugar. Examples of sugars includes butnot limited to sucrose, lactose, dextrose, trehalose, maltose, and thelike. The percentage of sugar may range from 5% to about 25%. Theresulting suspension can be homogenized or sonicated to reduce theparticle size. In some embodiments, the hydrated suspension is filteredthrough suitable filters to control the size distribution of the formedcomplexes. In some composition, the hydrated suspension can belyophilized to obtain the composition in powder form. In someembodiments, the hydrated composition can be autoclaved.

In some embodiments, the method of preparation of present inventioncomprising mixing active compound (for example, Docetaxel), cholesterylderivative (for example, cholesteryl sulfate) and phosphatidylcholinesuch as soy phosphatidylcholine or hydrogenated soy phosphatidylcholinein water or buffer. The resulting suspension can be homogenized orsonicated at any desired temperature ranging from 20-120° C. Examples ofbase or buffer includes but not limited to sodium succinate dibasic,sodium acetate sodium phosphate monobasic, sodium phosphate dibasic,sodium phosphate tribasic, sodium hydroxide, and the like. Thecomposition may further contain sugar. Examples of sugars includes butnot limited to sucrose, lactose, dextrose, trehalose, maltose, and thelike. The percentage of sugar may range from 5% to about 25%. Theresulting suspension can be homogenized or sonicated to reduce theparticle size. In some embodiments, the hydrated suspension is filteredthrough suitable filters to control the size distribution of the formedcomplexes. In some composition, the hydrated suspension can belyophilized to obtain the composition in powder form. In someembodiments, the hydrated composition can be autoclaved.

In some embodiments, the method of preparation of present inventioncomprising mixing active compound (for example, Paclitaxel), cholesterylderivative (for example, cholesteryl sulfate) and phosphatidylcholinesuch as soy phosphatidylcholine or hydrogenated soy phosphatidylcholinein water or buffer. The resulting suspension can be homogenized orsonicated at any desired temperature ranging from 20-120° C. Examples ofbase or buffer includes but not limited to sodium succinate dibasic,sodium acetate, sodium phosphate monobasic, sodium phosphate dibasic,sodium phosphate tribasic, sodium hydroxide, and the like. Thecomposition may further contain sugar. Examples of sugars includes butnot limited to sucrose, lactose, dextrose, trehalose, maltose, and thelike. The percentage of sugar may range from 5% to about 25%. Theresulting suspension can be homogenized or sonicated to reduce theparticle size. In some embodiments, the hydrated suspension is filteredthrough suitable filters to control the size distribution of the formedcomplexes. In some composition, the hydrated suspension can belyophilized to obtain the composition in powder form. In someembodiments, the hydrated composition can be autoclaved.

In some embodiments, the pH of the composition of invention ranges fromabout 3 to about 11, preferably having a pH of about 3.5 to about 8, andmore preferably having a pH of about 4.0 to pH 8.0. In some embodiments,aqueous solutions having suitable pH are prepared from water havingappropriate amount of buffers dissolved in it. In some preferredembodiments, buffers comprise mixtures of monobasic sodium phosphate,dibasic sodium phosphate and tribasic sodium phosphate. In somepreferred embodiments, buffers comprise sodium carbonate, sodiumbicarbonate, sodium hydroxide, ammonium acetate, sodium succinate,sodium citrate, tris (hydroxy-methyl) aminoethane, sodium benzoate,sodium acetate, and the like.

In some embodiments, filters are used to obtain the desired size rangeof the complexes from the filtrate. For example, the complexes can beformed and thereafter filtered through a 5 micron filter to obtaincomplex having a diameter of about 5 micron or less. Alternatively, 1μm, 500 nm, 200 nm, 100 nm or other filters can be used to obtaincomplexes having diameters of about 1 μm, 500 nm, 200 nm, 100 nm or anysuitable size range, respectively.

In some embodiments, the composition of the present invention can besterilized by filtering through 0.22 μm or 0.45 μm filter under asepticconditions. In another embodiments, the composition of the presentinvention can be sterilized by autoclaving in the range of 120° C.-130°C. for a duration of 15-20 minutes.

In some embodiments, the active compound-lipid complex (for example,amphotericin B-lipid complex) with or without deoxycholate is dried,e.g., by evaporation or lyophilization. In certain embodiments of theinvention, the active compound-lipid complex (for example, amphotericinB-lipid complex) with or without deoxycholate is lyophilized with one ormore cryoprotectants, such as sugars. Examples of sugars that find usein the present invention include but are not limited to trehalose,maltose, lactose, sucrose, glucose, and dextran. In preferredembodiments, the compositions of the present invention comprisetrehalose and/or sucrose. The lyophilization is generally accomplishedunder vacuum and can take place either with or without prior freezing ofthe active compound-lipid complex (for example, amphotericin B-lipidpreparation) with or without deoxycholate. While not limiting thelyophilization of the present invention to any particular configuration,the lyophilization in the present invention can be done, e.g., in vialsor other containers having desired volumes. The lyophilization can alsobe done as bulk in trays. When desired, the complexes can be resuspendedin any desirable solvent including water, saline, dextrose and buffer.

Pharmaceutical preparations that find use in the present inventioninclude but are not limited to tablets, capsules, pills, dragees,suppositories, solutions, suspensions, emulsions, ointments; gels can besuitable pharmaceutical preparations. In some embodiments, e.g., for theoral mode of administration, active compound-lipid complex (for example,amphotericin B-lipid complex, tacrolimus lipid complex, paclitaxel ordocetaxel lipid complexes) with or without deoxycholate is used in theform of tablets, capsules, lozenges, powders, syrups, aqueous solutions,suspensions and the like. In some embodiments, e.g., for topicalapplication and suppositories, active compound-lipid complex (forexample, amphotericin B-lipid complex, with or without deoxycholate) isprovided in the form of gels, oils, and emulsions, such as are known bythe addition of suitable water-soluble or water-insoluble excipients,for example polyethylene glycols, certain fats, and esters, compoundshaving a higher content of polyunsaturated fatty acids and derivativesthereof. Derivatives include but are not limited to mono-, di-, andtriglycerides and their aliphatic esters (for example, fish oils,vegetable oils etc.) or mixtures of these substances. In someembodiments, excipients that find use in conjunction with thecompositions of the present invention comprise those in which the drugcomplexes are sufficiently stable to allow for therapeutic use.

In some embodiments, preparations of active compound-lipid complex (forexample, amphotericin B-lipid complex with or without deoxycholate ortacrolimus-lipid complex, paclitaxel or docetaxel lipid complexes) areprepared in enteric coated tablets or capsules, e.g., to protect it fromacids in the stomach. “Enteric” refers to the small intestine, therefore“enteric coating” generally refers to a coating that substantiallyprevents release of a medication before it reaches the small intestine.While not limiting the invention to any particular mechanism of action,it is understood that most enteric coatings work by presenting a surfacethat is stable at acidic pH but breaks down rapidly at higher pH.Enteric coatings that find use in the present invention comprisecapsules filled with active compound-lipid complex (for example,amphotericin B-lipid complex with or without deoxycholate, tacrolimuslipid complex, paclitaxel or docetaxel lipid complexes) as according tomethods well known in the art.

Preparations of active compound-lipid complex (for example, amphotericinB-lipid complex) with or without deoxycholate of the present inventioncan comprise complexes of varying size, or can comprise complexes ofsubstantially uniform size. For example, in some embodiments thecomplexes have a size range of about 1 mm or less, while in preferredembodiments, the complexes are in the micron or sub-micron range. Insome embodiments, the complexes have a diameter of about 5 μm or less,such as 0.2 μm or less, or even 0.1 μm or less.

Active compound-lipid complex (for example, amphotericin B-lipidcomplex, with or without deoxycholate) of the present invention maycomprise or consist essentially of micelles, mixed micelles, liposomesand vesicles of different shape and sizes.

As noted above, the technology outlined in the present invention for thepreparation of amphotericin B complexes is also suitable for use withany other water-insoluble drugs.

In some embodiments, the inventive amphotericin B-lipid complex (with orwithout deoxycholate) is employed to treat a fungal infection, e.g., ina mammal. In this regard, the invention provides a method of treatingfungal infections comprising administering to a subject (e.g. a patienthaving a fungal infection) a composition comprising a complex ofamphotericin B-with or without deoxycholate and lipid(s) in an amountsufficient to treat the fungal infection within the subject.

The composition of the present invention can be employed to treatinfections caused by numerous fungi and parasites, including but notlimited to, Acremonium sp., Aspergillus fumigatus, Aspergilluspneumonia, Blastomyces dermatitides, Candida albicans, Candidaguillermondi, Candida tropicalis, Coccidioides immitis, Cryptococcusneoformans, Fusarium sp., Histoplasma capsulatum, Mucor mucedo,Rhodotorula sp., Sporothrix schenckii, Acanthamoeba polyphaga,Entomophthora sp., Histoplasma capsulatumm Leishmania brasiliensis,Rhizopus sp., Rhodotorula sp., Torulopsis glabrata, Paracoccidioidesbrasiliensis. Additional fungal pathogens include Trichosporon, Muco,Alternaria, Bipolaris, Curvularia, etc.

The composition of present invention can also be employed to treatVisceral Leishmaniasis also called as Kala-azar and infections caused byLeishmania donovani complex, L. d donovani, L. d infantum, L. darchibaldi, L. d chagasi, Phlebotomus sp. and Lutzomya logipalpis.

The composition of present invention can also be employed to treat viralinfections such as those caused, e.g., by human immunodeficiency virus(HIV), herpes simplex viruses (HSV-1 and HSV2), hepatitis C virus (HCV)and cyotomegalovirus (CMV).

In some embodiments, the inventive active compound lipid-complex (forexample, docetaxel-lipid complex or paclitaxel-lipid complex) isemployed to treat a cancer, e.g., in a mammal. In this regard, theinvention provides a method of treating cancer comprising administeringto a subject (e.g. a patient having a cancer) a composition comprising acomplex of active compound lipid-complex (for example, docetaxel-lipidcomplex or paclitaxel-lipid complex) and lipid(s) in an amountsufficient to treat the cancer within the subject. The cancer can be anytype of cancer in a mammal. Examples include, but are not limited tocancers of the head, neck, brain, blood, (e.g. leukemia, acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, lymphoma,myeloma), breast, lung, pancreas, bone, spleen, bladder, prostate,testes, colon, kidney, ovary and skin (e.g. Kaposi's sarcoma), bonemarrow, liver, stomach, tongue, mouth and larynx. In addition, activecompound-lipid complex of the present invention are useful in reducingthe tendency of cancer cells to develop a resistance to othertherapeutic agents such as anti-cancer agents, chemotherapy andradiation. Thus, other therapeutic agents can be advantageously employedwith the present invention in the formation of an active combination orby separate administration.

In some embodiments, the inventive active compound lipid-complex (forexample, tacrolimus-lipid complex) is employed to treat rejectionreactions caused by organ transplantations and can be administered organor tissue transplantation, e.g., in a mammal. In this regard, theinvention provides a method of preventing organ or tissue rejectioncomprising administering to a subject (e.g. a patient having an organ ortissue transplantation) a composition comprising a complex of activecompound lipid-complex (for example, tacrolimus-lipid complex) andlipid(s) in an amount sufficient to prevent an organ or tissue rejectionwithin the subject.

The examples of the present invention are illustrated below but theinvention is not limited to the following examples and modifications canbe made without departing from the purports described in thisapplication.

Example 1

Amphotericin B (1 gm) was suspended in aqueous medium at pH 1.5 to 3.5and mixed with 3 gm of Sodium Cholesteryl Sulfate. SoyaPhosphatidylcholine (7 gm) was stirred and mixed with Amphotericin B andSodium Cholesteryl Sulfate Complex for 30 min. The mixture was thensubjected to high pressure homogenization. The formulation waslyophilized in the presence of 7.5-9.5% sucrose and reconstituted inwater for injection. The particle size was determined using Nicompparticle sizer 380. The mean volume diameter amounted to less than 200nm.

Example 2

Amphotericin B formulation with lipids as described in Example I wasused to test the hemolysis of red blood cells (RBCs). At 0.16 mg/mLFUNGIZONE50% of the cells were lysed compared to Amphotericin B lipidsuspension where no lysis occurred after incubation with RBCs. Toxicitystudy was also carried out in Balb/c mice. A total of 9 mice (7 weeksold) were subjected to intravenous administration of amphotericin Bformulation at 20 mg/kg. The mice were monitored for 30 days. At the endof 30 days no mortality was observed. This indicated that maximumtolerated dose using this formulation exceeds 20 mg/kg.

Group Dose Survival I 20 mg/kg 9/9

Example 3

Amphotericin B (1 gm) was suspended in aqueous medium at pH 1.5 to 3.5and mixed with 3 gm of sodium cholesteryl sulfate. Hydrogenated soyaphosphatidylcholine (7 gm) was stirred and mixed with amphotericin B andsodium cholesteryl sulfate complex for 30 min. The mixture was thensubjected to high pressure homogenization. The formulation waslyophilized in the presence of 7.5% sucrose and reconstituted in waterfor injection. The particle size was determined using Nicomp particlesizer 380. The particle size was determined using Nicomp particle sizer380. The mean volume weighting diameter amounted to less than 200 nm

Example 4

Amphotericin B (20 mg) and sodium deoxycholate (6.56 mg) were dissolvedin water (10 mL) at pH 11.00 to 12.5 using sodium hydroxide. The pH wasthen adjusted to pH 7.00-8.5 with suitable acid (for example, phosphoricacid). Hydrogenated soy phosphatidylcholine (930 mg) and cholesterylsulfate (10.4 mg) was mixed in water (10 mL) and homogenized orsonicated for 30 minutes. The lipid suspension was then mixed withamphotericin B-deoxycholate solution and further homogenized orsonicated for 1 hr. The suspension can be heated if desired attemperature ranging from 25° C. to 60° C. The formulation waslyophilized in the presence of 7.5% sucrose and reconstituted in waterfor injection. The formulation was tested for toxicity in Balb/c miceand compared with Deoxycholate formulation of Amphotericin B(FUNGIZONE). The animals were weighed and assigned to different groupsrandomly (5 animals/group). The results are reported in the table belowas the number of mice surviving per total.

Treatment Dose (mg/kg) Survival/Total Fungizone ® 0.5 5/5 1.0 5/5 2.04/5 4.0 0/5 Amphotericin—B 12.0 5/5 Formulation 14.0 5/5 17.0 5/5 20.00/5The data indicated that the liposome formulation of amphotericin B wassignificantly less toxic when compared to the marketed product(FUNGIZONE).

Example 5

Amphotericin B formulation with lipids as described in Example IV wasprepared without deoxycholate. The resulting formulation was lyophilizedin the presence of 7.5% sucrose or lactose. This formulation also showedsimilar characteristics as of Example 4.

Example 6

Amphotericin B (50 mg) and Cholesteryl sulfate (50 mg) were mixedtogether in water at pH 2.5-3. SPC (500 mg) was suspended in waterseparately which was mixed with amphotericin B and cholesteryl sulfatesuspension and homogenized using high pressure homogenizer. Theformulation was lyophilized in the presence of 7.5% sucrose andreconstituted in water for injection. The reconstituted formulation wastested for toxicity in Balb/c mice with single dose intravenousinjection and no mortality was observed at 20 mg/kg dose level as foundin Example II. The particle size was determined using Nicomp particlesizer 380. The particle size data is given in the table below.

Particle Size Mean/Distributions (Volume Weighting) Mean VolumeWeighting 128.4 nm Diameter 99% Distribution 401.8 nm 90% Distribution224.6 nm 80% Distribution 175.9 nm 75% Distribution 160.3 nm 50%Distribution 110.2 nm 25% Distribution  75.9 nm

Example 7

Amphotericin B (100 mg) and deoxycholate (33 mg) were dissolved in waterat pH 9-12.00 and later adjusted to pH 7.5. The amphotericin Bsuspension was then mixed with cholesteryl sulfate (52 mg) andhydrogenated soyphosphatidylcholine (4.62 g) in water and sonicated at60 minutes. The formulation was lyophilized both in vials and in bulk inthe presence of 7.5% sucrose and reconstituted in water for injection.The particle size was determined using Nicomp particle sizer 380. Themean volume weighting diameter amounted to less than 200 nm

Particle Size Mean/Distributions (Volume Weighting) Mean VolumeWeighting 76.1 nm Diameter 99% Distribution 227.1 nm  90% Distribution130.2 nm  80% Distribution 103.1 nm  75% Distribution 94.3 nm 50%Distribution 66.0 nm 25% Distribution 46.2 nm

Example 8

Amphotericin B (50 mg) and Cholesteryl sulfate (50 mg) are mixedtogether in sodium succinate buffer at pH 2.5-3. SPC (500 mg) in sodiumsuccinate buffer is suspended in water separately which is mixed withamphotericin B and cholesteryl sulfate suspension and homogenized usinghigh pressure homogenizer. The formulation is lyophilized in thepresence of 7.5-9.5% sucrose or 9.5% lactose and reconstituted in waterfor injection. The particle size was determined using Nicomp particlesizer 380. The mean volume weighting diameter amounted to less than 200nm

Example 9

Amphotericin B (2 g) and Cholesteryl sulfate (1.04 g) were mixedtogether in succinate buffer at pH 2.5 and sonicated for 5 min at roomtemperature. Soy lecithin (18.96 g) in sodium succinate buffer (pH 2.5)was with Amphotericin-Cholesteryl sulfate suspension and homogenizedusing high pressure homogenizer. The formulation was then autoclaved at121° C. for 15 minutes before it was mixed with 7.5-9.5% sucrose or 9.5%lactose solution under aseptic conditions. The particle size wasdetermined using Nicomp particle sizer 380. The particle size data isshown in the table below.

Particle Size Mean/Distributions (Volume Weighting) Mean VolumeWeighting 693.3 nm Diameter 99% Distribution 1992.5 nm  90% Distribution1169.7 nm  80% Distribution 934.6 nm 75% Distribution 858.2 nm 50%Distribution 608.4 nm 25% Distribution 431.3 nmThe HPLC analysis of the inventive formulation comprising amphotericinB, soy phosphatidylcholine, cholesteryl sulfate was done and the resultsare outlined in the table below.

Components Assay Results Amphotericin B 96.4% Cholesteryl Sulfate 95.0%Soy Phosphatidylcholine 87.5%

Systemic Adverse Events:

A comparison between FUNGIZONE and Amphotericin B Lipid Suspension inHealthy Human Volunteers.

The safety and tolerance of FUNGIZONE versus Amphotericin B LipidSuspension was evaluated in Human male subjects. In this study a total24 volunteers were enrolled. Out of this six (n=6) were given FUNGIZONE(0.6 mg/kg) intravenously and eighteen (n=18) of them receivedAmphotericin B Lipid Suspension (0.6 mg/kg-1.5 mg/kg).

In the Amphotericin B Lipid Suspension, mild adverse events werereported in 3/18 (17%) healthy male subjects and 4/6 (66%) who wereinfused FUNGIZONE. Overall, Amphotericin B Lipid Suspension isapparently safe and well tolerated upto 1.5 mg/kg.

Example 10

Tacrolimus (20 mg) and Cholesteryl sulfate (20 mg) were mixed in water(10 mL) and sonicated for 30 min to form a suspension. SPC in water (10mL) was mixed with Tacrolimus and Cholesteryl Sulfate suspension andhomogenized using high pressure homogenizer. The formulation waslyophilized both in vials and in bulk in the presence of 7.5% sucroseand reconstituted in water for injection. The particle size wasdetermined using Nicomp particle sizer 380. The mean volume diameteramounted to less than 200 nm.

Example 11

Deoxycholate (1 mg) and Cholesteryl sulfate (1 mg) were mixed in waterand sonicated for 30 min to form a suspension. SPC in water was mixedwith Tacrolimus and Cholesteryl Sulfate suspension and homogenized usinghigh pressure homogenizer. The formulation was lyophilized in thepresence of 7.5-% sucrose and reconstituted in water for injection. Theparticle size was determined using Nicomp particle sizer 380. The meanvolume diameter amounted to less than 200 nm.

Example 12

Tacrolimus (100 mg), Cholesteryl sulfate (60 mg), and Soy lecithin (3.94g) were mixed together in water (70 mL) and homogenized using highpressure homogenizer. The resulting suspension was then filtered through0.2μ filter and then mixed with 7.5% sucrose solution (30 mL) andlyophilized both in vials and in bulk. The particle size was determinedusing Nicomp particle sizer 380. The mean volume weighting diameteramounted to less than 200 nm.

Particle Size Mean/Distributions (Volume Weighting) Mean VolumeWeighting 42.9 nm Diameter 99% Distribution 141.0 nm  90% Distribution76.5 nm 80% Distribution 59.2 nm 75% Distribution 53.8 nm 50%Distribution 36.5 nm 25% Distribution 25.2 nm

Example 13

Tacrolimus (200 mg), Cholesteryl sulfate (120 mg), and Soy lecithin(7.88 g) were mixed together in water (70 mL) and homogenized using highpressure homogenizer. The resulting suspension was then filtered through0.2μ filter and then mixed with 7.5% sucrose (30 mL) and lyophilizedboth in vials and in bulk. The particle size was determined using Nicompparticle sizer 380. The mean volume weighting diameter amounted to lessthan 200 nm.

Particle Size Mean/Distributions (Volume Weighting) Mean VolumeWeighting 76.4 nm Diameter 99% Distribution 240.8 nm  90% Distribution134.3 nm  80% Distribution 105.1 nm  75% Distribution 95.8 nm 50%Distribution 65.9 nm 25% Distribution 45.5 nm

The Tacrolimus lipid suspension was tested for toxicity in Balb/c mice.The single test dose at 10 mg/kg and 20 mg/kg was intravenouslyadministered to mice. All the mice survived with no significant loss ofbody weight. Similarly, repeat dose toxicity study was conducted with adose of 10 mg/kg or 20 mg/kg for consecutively 5 days with accumulateddose of 50 mg/kg and 100 mg/kg respectively. All the animals in thegroup survived. The results are reported in the table below as thenumber of mice surviving per total.

Treatment Dose (mg/kg) Survival/Total Single dose 10 5/5 20 5/5 Repeatdose 10 5/5 20 5/5

Example 14

Cholesteryl sulfate (2.08 mg) and hydrogenated soyphosphatidylcholine(185.92 mg) in 0.9% aq. Sodium chloride solution (2 mL) was sonicated at65° C. for 30 minutes before Doxorubicin (40 mg) in 0.9% sodium chloridesolution (2 mL) was added and further sonicated for 60 minutes. Theformulation was lyophilized in the presence of 7.5% sucrose or andreconstituted in water for injection.

Example 15

Cholesteryl sulfate (20 mg) and soy lecithin (156.8 mg) in 0.9% aq.sodium chloride solution was sonicated at 65° C. for 30 minutes beforeDoxorubicin (40 mg) in 0.9% sodium chloride solution (10 mL) was addedand further sonicated for 60 minutes. The formulation is lyophilized inthe presence of 7.5% sucrose and reconstituted in water for injection.The particle size was determined using Nicomp particle sizer 380. Themean volume diameter amounted to less than 200 nm.

Example 16

Docetaxel (20 mg), Cholesteryl sulfate (12.0 mg), and Soy lecithin (788mg) were mixed together in water (10 mL) and using high pressurehomogenizer. The formulation is lyophilized in the presence of 7.5%sucrose and reconstituted in water for injection. The particle size wasdetermined using Nicomp particle sizer 380. The mean volume weightingdiameter amounted to less than 200 nm.

Particle Size Mean/Distributions (Volume Weighting) Mean VolumeWeighting  93.9 nm Diameter 99% Distribution 264.1 nm 90% Distribution157.0 nm 80% Distribution 126.1 nm 75% Distribution 116.0 nm 50%Distribution  83.0 nm 25% Distribution  59.4 nm

Example 17

Docetaxel (40 mg), Cholesteryl sulfate (24.0 mg), and Soy lecithin (1.57g) were mixed together in water (10 mL) using high pressure homogenizer.The formulation is lyophilized in the presence of 7.5% sucrose andreconstituted in water for injection. The particle size was determinedusing Nicomp particle sizer 380. The mean volume diameter amounted toless than 200 nm

Example 18

Paclitaxel (20 mg), Cholesteryl sulfate (11.4 mg), and Soy lecithin(788.6 mg) were mixed together in water (10 mL) and homogenized usinghigh pressure homogenizer. The formulation is lyophilized in thepresence of 7.5% sucrose and reconstituted in water for injection. Theparticle size was determined using Nicomp particle sizer 380. The meanvolume weighting diameter amounted to less than 200 nm

Particle Size Mean/Distributions (Volume Weighting) Mean VolumeWeighting 124.1 nm Diameter 99% Distribution 357.4 nm 90% Distribution209.6 nm 80% Distribution 167.4 nm 75% Distribution 153.7 nm 50%Distribution 108.9 nm 25% Distribution  77.2 nm

The paclitaxel lipid suspension was tested for toxicity in Balb/c mice.The test dose (40 mg/kg) was intravenously administered to mice and theanimals were monitored for 30 days. All the mice survived with nosignificant loss of body weight. Similarly, repeat dose toxicity studywas conducted with a dose of 40 mg/kg for consecutively 5 days withaccumulated dose of 200 mg/kg. All the animals in the group survived.The study was monitored for 30 days. The results are reported in thetable below as the number of mice surviving per total

Treatment Dose (mg/kg) Survival/Total Single dose 40 3/3 Repeat dose 404/4

Example 19

Paclitaxel (40 mg), Cholesteryl sulfate (22.8 mg), and Soy lecithin(1.58 g) were mixed together in water (10 mL) and homogenized using highpressure homogenizer. The formulation is lyophilized in the presence of7.5% sucrose and reconstituted in water for injection. The particle sizewas determined using Nicomp particle sizer 380. The particle size datais shown in the table below.

Particle Size Mean/Distributions (Volume Weighting) Mean VolumeWeighting  839.1 nm Diameter 99% Distribution 3425.8 nm 90% Distribution1636.3 nm 80% Distribution 1185.7 nm 75% Distribution 1048.7 nm 50%Distribution  638.5 nm 25% Distribution  388.7 nm

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All references, including publications, patent applications, and patentscited herein, including those in the preceding list and otherwise citedin this specification, are hereby incorporated by reference to the sameextent as if each reference was individually and specifically indicatedto be incorporated by reference and were set forth in the entirelyherein.

Preferred embodiments of this invention are described, including thebest mode known to the inventors for carrying out the invention. Variousmodifications and variations of the described methods and systems of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention, and the inventors intend forthe inventions to be practiced otherwise than specifically describedherein. Accordingly, this invention includes all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context. Indeed, any modifications ofthe described modes for carrying out the invention that are obvious tothose skilled in the relevant fields are intended to be within the scopeof the following claims.

What is claimed is:
 1. A method of treating a disease in a subject,comprising: a) using an aqueous system to prepare a compositioncomprising a complex, said complex comprising at least one activecompound and at least one lipid; and b) administering said compositionto a subject.
 2. The method of claim 1, wherein said complex comprises alipid compound suspension and wherein said aqueous system comprises aprocess comprising: a) preparing a suspension comprising said at leastone active compound and said at least one lipid in a first aqueousmedium at a pH between about pH 4.0 and pH 8.0; b) treating saidsuspension to form a lipid-compound suspension of defined particle size;c) lyophilizing the lipid-compound suspension of defined particle sizeto form lyophilized material; and d) reconstituting said lyophilizedmaterial with a second aqueous medium to obtain a suspension of lipidformulation of defined particle size, said defined particle size havinga mean particle size of less than 5 microns.
 3. The method of claim 1,wherein said at least one active compound is selected from the groupconsisting of amphotericin-B with deoxycholate, amphotericin B withoutdeoxycholate, docetaxel, paclitaxel, tacrolimus, doxorubicin,Epirubicin, anthracyclines, and etoposide.
 4. The method of claim 1,wherein said at least one lipid is selected from the group consisting ofegg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), soyphosphatidylcholine (SPC), hydrogenated soy phosphatidylcholine (HSPC),dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol(DMPG), dipalmitoylphosohatidylcholine (DPPC),disteroylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol(DMPG), cholesterol (Chol), cholesterol sulfate and its salts (CS),cholesterol hemisuccinate and its salts (Chems), cholesterol phosphateand its salts (CP), cholesterylphosphocholine and otherhydroxycholesterol or amino cholesterol derivatives, cholesterylsuccinate, cholesteryl oleate, polyethylene glycol derivatives ofcholesterol (cholesterol-PEG), coprostanol, cholestanol, cholestane,cholic acid, cortisol, corticosterone, hydrocortisone, and calciferol,monoglycerides, diglycerides, triglycerides, carbohydrate-based lipidsselected from a group consisting of galactolipid, mannolipid,galactolecithin, β-sitosterol, stigmasterol, stigmastanol, lanosterol,α-spinasterol, lathosterol, campesterol, phosphatidylcholine,phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine,phosphatdylinositol, phosphatidic acid, and pegylated derivatives ofdistearoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol,dimyristoylphosphatidylglycerol, and dioleoylphosphatidylglycerol. 5.The method of claim 1, wherein said at least one lipid comprises one ormore of fatty acids selected from a group consisting of saturated orunsaturated fatty acids.
 6. The method of claim 1, wherein saidcomposition further comprises polyethylene glycol.
 7. The method ofclaim 1, wherein said at least one lipid is selected from the groupconsisting of cholesterol or cholesterol sulfate and salts thereof,cholesterol hemisuccinate and salts thereof, cholesterol phosphate andsalts thereof, and wherein said composition further comprises at leastone phospholipid.
 8. The method of claim 1, wherein said at least onelipid comprises a cholesterol or cholesterol derivative, wherein themole ratio of active compound to cholesterol or cholesterol derivativeis between about 1:1 and 1:10.
 9. The method of claim 1, wherein said atleast one lipid comprises hydrogenated soy phosphatidylcholine or soyphosphatidylcholine, wherein the mole ratio of active compound andhydrogenated soy phosphatidylcholine or soy phosphatidylcholine isbetween about 1:1 to about 1:90.
 10. The method of claim 1, wherein saidcomposition comprises active compound at a concentration of from about0.5 mg/mL to about 25 mg/mL.
 11. The method of claim 1, wherein saidcomposition comprises a total lipid concentration of from 2.5% by weightto about 95% by weight.
 12. The method of claim 1, wherein the molarratio of active compound to lipid in said composition is between 1:10 to1:100.
 13. The method of claim 1, wherein the weight-to-weight ratio oftotal active compound to total lipid in said composition is between 1:10to 1:60.
 14. The method of claim 1, wherein said composition comprises aform selected from the group consisting of powder, solution, suspension,emulsion, micelle, liposome, lipidic particle, gel, and paste form. 15.The method of claim 14, wherein said composition comprises a pluralityof micelles, wherein said micelles are in the form of monomeric,dimeric, polymeric or mixture of micelles and vesicles.
 16. The methodof claim 1, wherein said preparing of a composition comprising a complexcomprises preparing said complex in a lyophilized form.
 17. The methodof claim 16, wherein said preparing said complex in a lyophilized formcomprises using a cryoprotectant, wherein said cryoprotectant comprisesone or more sugars selected from a group consisting of trehalose,maltose, lactose, sucrose, glucose, and dextran.
 18. The method of claim1, wherein, said composition comprises a tablet or a filled capsule, andoptionally comprises an enteric coating material.
 19. The method ofclaim 1, wherein said active compound is a partially water soluble orwater insoluble drug.
 20. The method of claim 1, wherein saidadministering comprises oral, intravenous, subcutaneous, parenteral,intraperitoneal, rectal, vaginal, and/or topical delivery of saidlipidic composition to said subject.
 21. A process for preparing a lipidformulation of an active compound, wherein said process comprises usingan aqueous system to prepare a composition comprising a complex, saidcomplex comprising at least one active compound and at least one lipid.22. The process of claim 21, wherein said process is a process forpreparing a lipid formulation of defined particle size, wherein saidprocess comprises: a) preparing a suspension comprising at least oneactive compound and at least one lipid in a first aqueous medium at a pHbetween about pH 4.0 and pH 8.0; b) treating said suspension to form alipid-compound suspension of defined particle size; c) lyophilizing thelipid-compound suspension of defined particle size to form lyophilizedmaterial; and d) reconstituting said lyophilized material with a secondaqueous medium to obtain a suspension of lipid formulation of definedparticle size, said defined particle size having a mean particle size ofless than 5 microns.
 23. The process of claim 22, wherein said firstaqueous medium is water.
 24. The process of claim 22, wherein said firstaqueous medium and said second aqueous medium are different.
 25. Theprocess of claim 22, wherein said treating said suspension comprisesextruding said suspension through a selected size aperture.
 26. Theprocess of claim 22, wherein said treating said suspension compriseshigh pressure split homogenization.
 27. The process of claim 22 whereinsaid lyophilizing is in the presence of a cryoprotectant.
 28. Theprocess of claim 21, wherein said active compound comprises an activecompound selected from the group consisting of a polyene antibiotic, amacrolide, an anti-cancer drug, and an immunosuppressant.
 29. Theprocess of claim 21, wherein said active compound comprises a compoundselected from the group consisting of docetaxel, paclitaxel,doxorubicin, epirubicin, tamoxifen, endoxifen, etoposide,anthracyclines, amphotericin B, tacrolimus, and sacrolimus.
 30. Theprocess of claim 21, wherein said at least one lipid is selected fromthe group consisting of egg phosphatidylcholine, eggphosphatidylglycerol, soy phosphatidylcholine, hydrogenated soyphosphatidylcholine, dimyristoylphosphatidylcholine,dimyristoylphosphatidylglycerol, dipalmitoylphosohatidylcholine,disteroylphosphatidylglycerol, dipalmitoylphosphatidylglycerol,cholesterol, cholesterol sulfate and its salts, cholesterolhemisuccinate and its salts, cholesterol phosphate and its salts,cholesterylphosphocholine and other hydroxycholesterol or aminocholesterol derivatives, cholesteryl succinate, cholesteryl oleate,polyethylene glycol derivatives of cholesterol (cholesterol-PEG),coprostanol, cholestanol, cholestane, cholic acid, cortisol,corticosterone, hydrocortisone, and calciferol.
 31. The process of claim21, wherein said lipid formulation comprises cholesterol sulfate, andwherein the molar ratio of active compound to cholesterol sulfate insaid suspension is in between about 1:1 to about 1:10.
 32. The processof claim 22, wherein the composition mean particle size uponreconstitution is about 10-5000 nm.
 33. The process of claim 21, whereinsaid at least one active compound exhibits poor solubility in water,alcohols, and halogenated hydrocarbon solvents.
 34. The process of claim22, wherein said suspension of lipid formulation of defined particlesize comprises a suspension of liposomes and/or lipidic particles.
 35. Amethod treating a cell with a lipidic composition comprising at leastone active agent and at least one lipid, comprising: a) using an aqueoussystem to prepare a composition comprising a complex, said complexcomprising at least one active compound and at least one lipid; and b)exposing said cell to said lipidic composition.
 36. The method of claim35, wherein said exposing said cell comprises exposing said cell to saidlipidic composition in vivo.
 37. The method of claim 35, wherein saidsubject is a mammal.
 38. The method of claim 37, wherein said mammal ishuman.